Halohydrin dehalogenases and related polynucleotides

ABSTRACT

The present invention relates to novel halohydrin dehalogenase polypeptides and the polynucleotides that encode them. These polypeptides are useful in the production of 4-substituted-3-butyric acid derivatives and vicinal cyano, hydroxyl substituted carboxylic acid esters. The invention also provides related vectors, host cells and methods.

FIELD OF THE INVENTION

The present invention relates to novel halohydrin dehalogenasepolypeptides and the polynucleotides that encode them.

BACKGROUND OF THE INVENTION

Halohydrin dehalogenase (“HHDH”), also named halohydrinhydrogen-halide-lyase or halohydrin epoxidase, [EC4.5.1] catalyzes theinterconversion of 1,2-halohydrins and the corresponding 1,2-epoxides:

U.S. Pat. No. 4,284,723 describes the use of a halohydrin epoxidase forthe production of propylene oxide. U.S. Pat. Nos. 5,166,061 and5,210,031 describe the use of this enzyme activity for the conversion of1,3-dichloropropanol (DCP) and epichlorohydrin (ECH) respectively to4-chloro-3-hydroxybutyronitrile (CHBN). HHDH enzymes from Agrobacteriumradiobacter and Corynebacterium have been characterized on a broad rangeof halogenated substrates (Van Hylckama Vlieg et al., J. Bacteriol.(2001) 183:5058-5066; Nakamura et al., Appl. Environ. Microbiol. (1994)60:1297-1301; Nagasawa et al., Appl. Microbiol. Biotechnol. (1992)36:478-482).

HHDH also catalyzes the ring opening of epoxides with nucleophiles otherthan chloride or bromide. It has been demonstrated that azide (N₃ ⁻),nitrite (NO₂ ⁻) and cyanide (CN⁻) can replace chloride in the opening ofepoxides (see Nakamura et al., Biochem. Biophys Res. Comm. (1991)180:124-130; Nakamura et al., Tetrahedron (1994) 50: 11821-11826; LutjeSpelberg et al., Org. Lett. (2001) 3:41-43; Lutje Spelberg et al.,Tetrahedron Assym. (2002) 13:1083):

Nakamura et al. (Tetrahedron (1994) 50: 11821-11826) describe the use ofHHDH for the direct conversion of DCP to chloro-3-hydroxy-butyronitrile(CHBN) through epichlorohydrin (ECH) as the intermediate:

Some halohydrin dehalogenases have been characterized. For example, HHDHfrom A. radiobacter AD1 is a homotetramer of 28 kD subunits.Corynebacterium sp. N-1074 produces two HHDH enzymes, one of which iscomposed of 28 kD subunits (Ia), while the other is composed of relatedsubunits of 35 and/or 32 kD (Ib). HHDH from some sources is easilyinactivated under oxidizing conditions in a process that leads todissociation of the subunits, has a pH optimum from pH 8 to 9 and anoptimal temperature of 50° C. (Tang, Enz. Microbial Technol. (2002)30:251-258; Swanson, Curr. Opin. Biotechnol. (1999) 10:365-369). Theoptimal pH for HHDH catalyzed epoxide formation has been reported as 8.0to 9.0 and the optimal temperature in the range of from 45° C. to 55° C.(Van Hylckama Vlieg et al., J. Bacteriol. (2001) 183:5058-5066; Nakamuraet al., Appl. Environ. Microbiol. (1994) 60:1297-1301; Nagasawa et al.,Appl. Microbiol. Biotechnol. (1992) 36:478-482). The optimal pH for thereverse reaction, ring opening by chloride, has been reported for thetwo Corynebacterium sp. N-1074 enzymes and is 7.4 (Ia) or 5 (Ib). Sitedirected mutagenesis studies on the A. radiobacter AD1 HHDH indicatedthat oxidative inactivation is due to disruption of the quartenarystructure of the enzyme by oxidation of cysteine residues (Tang et al.,Enz. Microbial Technol. (2002) 30:251-258).

Purified HHDH enzymes from different sources exhibit specific activitieson DCP ranging from 146 U/mg (Ib) to 2.75 U/mg (Ia) (Nakamura et al.,Appl. Environ. Microbiol. 1994 60:1297-1301; Nagasawa et al., Appl.Microbiol. Biotechnol. (1992) 36:478-482). The high activity of the Ibenzyme is accompanied by a high enantioselectivity to produce R-ECH fromDCP, while the Ia enzyme produces racemic ECH.

HHDH encoding genes have been identified in Agrobacterium radiobacterAD1 (hheC), Agrobacterium tumefaciens (halB), Corynebacterium sp (hheAencoding Ia and hheB encoding Ib), Arthrobacter sp. (hheA_(AD2)), andMycobacterium sp. GP1 (hheB_(GP1)). All enzymes have been functionallyexpressed in E. coli.

It is highly desirable for commercial applications of HHDH that theenzyme exhibits high volumetric productivity, that reactions run tocompletion in a relatively short period of time, with a high finalproduct concentration, with high enanantioselectivity, and that nochemical side products are formed. These characteristics of a processcan generally be used to define the broad characteristics of the enzyme:low Km for the substrate(s), high process stability, high specificactivity, no substrate and product inhibition under conditions wherechemical reactions are not proceeding. Currently available HHDH enzymesdo not fulfill all of these criteria. For instance, the conversion on1,2-epoxybutane and cyanide to 3-hydroxyvaleronitrile by HHDH proceedsat a maximum rate of 3 mmol/hr and this rate is sustained for only 10minutes (Nakamura et al., Biochem. Biophys Res. Comm. (1991)180:124-130). Conversion of DCP and ECH to4-chloro-3-hydroxybutyro-nitrile (CHBN) is also limited to rates of 2-3mmol/hr (Nakamura, U.S. Pat. Nos. 5,166,061 and 5,210,031). An in depthanalysis of the ECH to CHBN conversion reveals that while the hheBencoded HHDH-Ib enzyme has high activity, high productivity ismaintained for only 20 min after which further conversion occurs at arate that is at least 50-fold slower, with the overall conversion atjust over 60% (Nakamura et al. Tetrahedron (1994) 50: 11821-11826). Thedirect conversion of DCP, via ECH to CHBN proceeds at a reduced rate andresults in a 65.3% yield. Thus, HHDH as described in the literature doesnot meet the desired criteria for a catalyst in commercial applications.

Accordingly, new halohydrin dehalogenases would be highly desirable.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a polypeptide, typically anisolated and optionally purified polypeptide (more typically, arecombinant polypeptide) having halohydrin dehalogenase activity,wherein the polypeptide comprises an amino acid sequence selected fromthe group consisting of:

(a) a polypeptide having an amino acid sequence that is at least 99%identical to SEQ ID NO: 4, 12, 16, 18, 34, 38, 44, 48, 52, 66, 80, 84,114, 154, 158, 170, or 270;(b) a polypeptide having an amino acid sequence that is at least 98%identical to SEQ ID NO: 10, 14, 68, 118, 164, 166, or 180;(c) a polypeptide having an amino acid sequence that is at least 97%identical to SEQ ID NO: 110, 162, 262, 422, 440 or 520;(d) a polypeptide having an amino acid sequence that is at least 96%identical to SEQ ID NO: 116 or 448;(e) a polypeptide having an amino acid sequence that is at least 95%identical to SEQ ID NO: 264, 266, 470 or 476;(f) a polypeptide having an amino acid sequence that is at least 93%identical to SEQ ID NO: 200;(g) a polypeptide having an amino acid sequence that is at least 89%identical to SEQ ID NO: 442;(h) a polypeptide having an amino acid sequence that is at least 88%identical to SEQ ID NO: 702;(i) a polypeptide that is at least 80% identical to SEQ ID NO: 2, whenoptimally aligned with SEQ ID NO: 2, and which comprises at least oneamino acid residue selected from the group consisting of T at (residue)position 2, A or P or S at position 3, V at position 4, D at position 6,either I or F at position 9, L at position 10, S at position 13, S atposition 14, K at position 15, C at position 16, T or R at position 17,either C or S or K at position 20, T at position 24, Q at position 26, Fat position 28, T at position 29, A at position 30, L at position 31, Gat position 33, R at position 34, L at position 35, N at position 36, Hat position 37, D at position 40, L at position 44, P at position 45,either P or A at position 47, N at position 52, V at position 54, R atposition 55, D at position 56, K at position 58, G or D at position 61,V at position 63, R at position 72, I at position 75, P at position 76,C at position 78, Y at position 82, either S or L at position 84, A atposition 85, E at position 91, D at position 93, Q or G at position 95,N at position 96, K at position 107, A at position 112, either T, S or Gat position 114, A at position 115, P at position 117, N at position120, E at position 121, P at position 122, R at position 126, V atposition 130, S at position 133, A or V at position 134, L, W or V atposition 136, H at position 139, I or R at position 142, S at position144, S at position 146, T at position 152, S at position 153, either Sor A at position 154, V at position 168, T at position 169, F atposition 177, V at position 178, I at position 180, G or I at position181, K at position 184, Y at position 186, L at position 194, N atposition 198, M at position 199, E at position 215, G at position 236, Vat position 237, L at position 238, T at position 240, either I or A orV at position 245, Y at position 249, V or I at position 252, and V atposition 254;(j) a polypeptide encoded by a nucleic acid that hybridizes understringent conditions over substantially the entire length of a nucleicacid corresponding to SEQ ID NO: 1, and wherein the encoded polypeptide,when optimally aligned with SEQ ID NO: 2, comprises an amino acidsequence having at least one amino acid residue selected from the groupconsisting of T at (residue) position 2, A, P or S at position 3, V atposition 4, D at position 6, either I or F at position 9, L at position10, S at position 13, S at position 14, K at position 15, C at position16, T or R at position 17, either S or K or C at position 20, T atposition 24, Q at position 26, F at position 28, T at position 29, A atposition 30, L at position 31, G at position 33, R at position 34, L atposition 35, N at position 36, H at position 37, D at position 40, L atposition 44, P at position 45, either P or A at position 47, N atposition 52, V at position 54, R at position 55, D at position 56, K atposition 58, G or D at position 61, V at position 63, R or Q at position72, I at position 75, P at position 76, C at position 78, Y at position82, either S or L at position 84, A at position 85, E at position 91, Dat position 93, Q or G at position 95, N at position 96, G at position99, K at position 107, A at position 112, either T, G or S at position114, A at position 115, P at position 117, N at position 120, E atposition 121, P at position 122, R at position 126, V at position 130, Sat position 133, A or V at position 134, L, W or V at position 136, H atposition 139, I or R at position 142, S at position 144, S at position146, T at position 152, S at position 153, either S or A at position154, V at position 168, T at position 169, F at position 177, V atposition 178, I at position 180, G at position 181, K at position 184, Yat position 186, Tat position 189, L at position 194, N at position 198,Mat position 199, E at position 215, A at position 222, G at position236, V at position 237, L at position 238, T at position 240, either Ior A or V at position 245, V or I at position 252, and V at position254.

In another aspect, the present invention is directed to a polypeptide,typically an isolated and optionally purified polypeptide (moretypically, a recombinant polypeptide) having HHDH , wherein thepolypeptide comprises an amino acid sequence selected from the groupconsisting of (a), (b), (c), (d), (e), (f), (g), (h), (i) and (j) asdescribed above, and further comprises an amino acid residue selectedfrom the group consisting of Q at position 37, Y at position 70, Q atposition 72, Q at position 80, G at position 99, R at position 107, T atposition 146, C at position 153, F at position 186, T at position 189,and A at position 222.

In another aspect, the present invention is directed to halohydrindehalogenases (HHDH) having from 1.4 fold to 10,000 fold greateractivity as compared to wild-type halohydrin dehalogenase fromAgrobacterium sp. (SEQ ID NO: 2).

In a further aspect, the present invention is directed to an isolated orrecombinant polypeptide having at least 1.4 fold greater HHDH activityas compared to wild-type HHDH having the amino acid sequence of SEQ IDNO: 2, and

wherein the polypeptide is encoded by a nucleic acid that hybridizesunder stringent conditions over substantially the entire length of anucleic acid having a sequence selected from the group consisting of SEQID NO: 3, 9, 11, 13, 15, 17, 33, 37, 43 , 47, 49, 51, 65, 67, 79, 83,109, 113, 115, 117, 153, 157, 161, 163, 165, 169, 179, 161, 199, 261,263, 265, 269, 421, 439, 441, 447, 469, 475, 519, 701, 725, 729, 731,733, 735, 737, and complementary sequences thereof.

In another aspect, the present invention is directed to HHDHpolynucleotides that encode polypeptides having halohydrin dehalogenaseactivity.

In a still further aspect, the present invention is directed to a vectorcomprising an HHDH polynucleotide of the present invention operativelylinked to a promoter.

In other embodiments, the present invention is directed to host cellsand methods for producing HHDH polypeptides of the present inventionfrom such host cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a 3944 by expression vector (PCK110700) of the presentinvention comprising a p15A origin of replication (P15A ori), a ladrepressor, a T5 promoter, a T7 ribosomal binding site (T7g10), and achloramphenicol resistance gene (camR).

FIG. 2 depicts the percent conversion vs. time for the reactions ofethyl (S)-4-chloro-3-hydroxybutyrate with aqueous hydrocyanic acid inthe presence of various halohydrin dehalogenase enzymes that aredescribed in Examples 8 through 12.

DETAILED DESCRIPTION HHDH Polypeptides

The present invention provides novel polypeptides having halohydrindehalogenase (“HHDH”) activity, as well as the polynucleotides thatencode them. The HHDH polypeptides of the present invention are suitablefor catalyzing the conversion of 4-halo-3-hydroxybutyric acidderivatives to 4-substituted-3-hydroxybutyric acid derivatives, using,for example, the methods described in the patent application entitled,“Enzymatic Processes for the Production of4-Substituted-3-Hydroxybutyric Acid Derivatives,” corresponding toAttorney Docket No. 0339.210US, filed on Aug. 11, 2003 and assigned U.S.Ser. No. 10/639,159, which is hereby incorporated herein by reference.These invention polypeptides are also suitable for catalyzing theconversion of vicinal halo, hydroxy substituted carboxylic acid estersto vicinal cyano, hydroxy substituted carboxylic acid esters using, forexample the methods described in the patent application entitled,“Enzymatic Processes for the Production of4-Substituted-3-Hydroxybutyric Acid Derivatives and Vicinal Cyano,Hydroxy Substituted Carboxylic Acid Esters,” corresponding to AttorneyDocket No. 0339.310US, filed on Feb. 18, 2004 and assigned U.S. Ser. No.10/782,258, which is hereby incorporated by reference. Polypeptides ofthe present invention are particularly useful as catalysts forconverting halohydrins to cyanohydrins, which are useful aspharmaceutical intermediates. In a specific application, HHDHpolypeptides of the present invention are used to catalyze theconversion of ethyl-4-chloro-3-hydroxybutyrate toethyl-4-cyano-3-hydroxybutyrate. Examples illustrating such conversionare provided hereinbelow. A more detailed description of such uses isprovided in the aforementioned patent applications entitled, “EnzymaticProcesses for the Production of 4-Substituted-3-Hydroxybutyric AcidDerivatives” and “Enzymatic Processes for the Production of4-Substituted-3-Hydroxybutyric Acid Derivatives and Vicinal Cyano,Hydroxy Substituted Carboxylic Acid Esters.” Id.

The present invention provides an isolated or recombinant polypeptidehaving HHDH activity, wherein the HHDH polypeptide comprises an aminoacid sequence selected from the group consisting of: a polypeptidehaving an amino acid sequence that is at least 99% identical to SEQ IDNO: 4, 12, 16, 18, 34, 38, 44, 48, 52, 66 80, 84, 114, 154, 158, 170, or270.

As used herein, the terms “HHDH activity” and “halohydrin dehalogenaseactivity” are used interchangeably herein to refer to the ability tocatalyze the conversion of ethyl (S)-4-chloro-3-hydroxybutyrate (“ECHB”)to a detectable amount of ethyl (R) 4-cyano-3-hydroxybutyrate (“HN”)using the assay described in Example 5A. The term “HHDH polypeptide”refers herein to a polypeptide having HHDH activity. The term “HHDHpolynucleotide” refers to a polynucleotide encoding a polypeptide havingHHDH activity.

As used herein, the term “isolated” refers to a nucleic acid,polynucleotide, polypeptide, protein, or other component that ispartially or completely separated from components with which it isnormally associated (other proteins, nucleic acids, cells, syntheticreagents, etc.). A nucleic acid or polypeptide is “recombinant” when itis artificial or engineered, or derived from an artificial or engineeredprotein or nucleic acid. For example, a polynucleotide that is insertedinto a vector or any other heterologous location, e.g., in a genome of arecombinant organism, such that it is not associated with nucleotidesequences that normally flank the polynucleotide as it is found innature is a recombinant polynucleotide. A protein expressed in vitro orin vivo from a recombinant polynucleotide is an example of a recombinantpolypeptide. Likewise, a polynucleotide sequence that does not appear innature, for example a variant of a naturally occurring gene, isrecombinant.

The terms “percent identity,” “% identity,” “percent identical,” and “%identical” are used interchangeably herein to refer to the percent aminoacid sequence identity that is obtained by ClustalW analysis (version W1.8 available from European Bioinformatics Institute, Cambridge, UK),counting the number of identical matches in the alignment and dividingsuch number of identical matches by the length of the referencesequence, and using the following default ClustalW parameters to achieveslow/accurate pairwise optimal alignments—Gap Open Penalty:10; GapExtension Penalty:0.10; Protein weight matrix: Gonnet series; DNA weightmatrix: IUB; Toggle Slow/Fast pairwise alignments=SLOW or FULLAlignment.

The present invention also provides a polypeptide having an amino acidsequence that is at least 98% identical to SEQ ID NO: 10, 14, 68, 118,164, 166, or 180. Desirable HHDH polypeptides include those that are atleast 99% identical to SEQ ID NO: 10, 14, 68, 118, 164, 166, or 180.

In another embodiment, the present invention provides a polypeptidehaving an amino acid sequence that is at least 97% identical to SEQ IDNO: 110, 162, 262, 422, 440, or 520. Some HHDH polypeptides of thepresent invention are at least 98%, and sometimes at least 99% identicalto SEQ ID NO: 110, 162, 262 422, 440, or 520.

In yet another embodiment, the present invention is directed to apolypeptide, typically an isolated and purified polypeptide having HHDHactivity greater than the wild-type HHDH of SEQ ID NO. 2, and having anamino acid sequence that is at least 93% identical to SEQ ID NO: 200,typically, 95% identical to SEQ ID NO: 200; more typically, 97%identical to SEQ ID NO: 200; most typically, 99% identical to SEQ ID NO:200.

In still another embodiment, the present invention is directed to apolypeptide, typically an isolated and purified polypeptide having HHDHactivity greater than the wild-type HHDH of SEQ ID NO. 2, and having anamino acid sequence that is at least 89% identical to SEQ ID NO: 442;typically, 93% identical to SEQ ID NO: 442; more typically, 95%identical to SEQ ID NO: 442; even more typically, 97% identical to SEQID NO: 442; most typically, 99% identical to SEQ ID NO: 442.

In another embodiment, the present invention is directed to apolypeptide, typically an isolated and purified polypeptide having HHDHactivity greater than the wild-type HHDH of SEQ ID NO. 2, and having anamino acid sequence that is at least 88% identical to SEQ ID NO: 702;typically, 93% identical to SEQ ID NO: 702: more typically, 95%identical to SEQ ID NO: 702; even more typically, 97% identical to SEQID NO: 702; most typically, 99% identical to SEQ ID NO: 702.

In a further embodiment, the present invention provides an HHDHpolypeptide having an amino acid sequence that is at least 96% identicalto SEQ ID NO: 116 or 448. HHDH polypeptides of the present inventioninclude those that are least 97% identical, 98% identical, and 99%identical to SEQ ID NO: 116 or 448.

The present invention further provides an HHDH polypeptide having anamino acid sequence that is at least 95% identical to SEQ ID NO: 264,266, 470 or 476. Desirable HHDH polypeptides of the present inventioninclude those that are least 96% identical, 97% identical, 98%identical, and 99% identical to SEQ ID NO: 264, 266, 470 or 476.

The present invention further provides an HHDH polypeptide that is atleast 80% identical to SEQ ID NO: 2, when optimally aligned with SEQ IDNO:2, and which further has one or more substitutions selected from thegroup consisting of S2T, either T3A or T3P, A4V, V6D, either V9I or V9F,K10L, G13S, G14S, M15K, G16C, either S17T or S17R, either R20S, R20C orR20K, A24T, H26Q, V28F, A29T, C30A, H31L, E33G, S34R, F35L, K36N, Q37H,E40D, F44L, A45P, either T47P or T47A, K52N, M54V, S55R, E56D, E58K,either E61G or E61D, 163V, Q72R, V75I, L76P, S78C, F82Y, either P84S orP84L, E85Q, K91E, A93D, E95Q or E95G, D96N, R107K, V112A, either A114Tor A114G or A1145, V115A, 5117P, K120N, K121E, R122P, H126R, I130V,A133S, T134A or T134V, F136L or F136W or F136V, W139H, L1421 or L142R,T144S, T146S, A152T, C153S, either T154S or T154A, I168V, P169T, Y177F,L178V, S1801, E181G or E181I, P184K, F186Y, T194I, H198N, V199M, K215E,V236G, F237V, W238L, A240T, either M245I or M245A or M245V, W249Y, M252Vor M252I, and E254V. In some embodiments, HHDH polypeptides of thepresent invention are at least 85% identical to SEQ ID NO: 2 and havingone or more of the substitutions indicated above. Some HHDH polypeptidesof the present invention are at least about 90% identical to SEQ ID NO:2, some are at least about 95% identical to SEQ ID NO: 2, and others areat least 99% identical to SEQ ID NO: 2, all having one or more of thesubstitutions indicated above. Some of these HHDH polypeptides have atleast 2 or more of the aforementioned substitutions, and some of theseHHDH polypeptides have at least 3 or more of the aforementionedsubstitutions.

When optimally aligned with sequence SEQ ID NO: 2, certain HHDHpolypeptides of the present invention have a sequence corresponding toSEQ ID NO: 2, but one or more amino acid substitutions selected from thegroup consisting of S2T, either T3A or T3P, A4V, V6D, either V9I or V9F,K10L, G135, G14S, M15K, G16C, either S17T or S17R, either R20S, R20C orR20K, A24T, H26Q, V28F, A29T, C30A, H31L, E33G, S34R, F35L, K36N, Q37H,E40D, F44L, A45P, either T47P or T47A, K52N, M54V, S55R, E56D, E58K,either E61G or E61D, 163V, Q72R, V75I, L76P, S78C, F82Y, either P84S orP84L, E85Q, K91E, A93D, E95Q or E95G, D96N, R107K, V112A, either A114Tor A114G or A114S, V115A, S117P, K120N, K121E, R122P, H126R, I130V,A133S, T134A or T134V, F136L or F136W or F136V, W139H, L1421 or L142R,T144S, T146S, A152T, C153S, either T154S or T154A, I168V, P169T, Y177F,L178V, S180I, E181G or E181I, P184K, F186Y, T194I, H198N, V199M, K215E,V236G, F237V, W238L, A240T, either M245I or M245A or M245V, W249Y, M252Vor M252I, and E254V. In some embodiments, the HHDH polypeptides have twoor more, and sometimes three or four or more of the aforementionedsubstitutions. Typically, in this embodiment, the resulting HHDHpolypeptide has at least 80% of sequence identity with SEQ ID NO: 2;more typically, at least 90% sequence identity; even more typically atleast 95% sequence identity; and yet even more typically at least 98%sequence identity.

The HHDH polypeptides described herein may further have one or moreamino acid residues selected from the group consisting of Q at position37, Y at position 70, Q at position 72, Q at position 80, G at position99, R at position 107, T at position 146, C at position 153, F atposition 186, T at position 189, and A at position 222. In someembodiments, the HHDH polypeptides of the present invention have two,three, or four or more of these selected residues. Of these residues,Q37, Y70, Q87, R107, T146, C153, and F186 appear to correlate favorablywith HHDH activity. Others appear to correlate favorably well withresistance to inhibition by ethyl-4-chloroacetate, as discussed in moredetail below.

Two sequences are “optimally aligned” when they are aligned forsimilarity scoring using a defined amino acid substitution matrix (e.g.,BLOSUM62), gap existence penalty and gap extension penalty so as toarrive at the highest core possible for that pair of sequences Aminoacid substitution matrices and their use in quantifying the similaritybetween two sequences are well-known in the art. See e.g., Dayhoff etal. (1978), “A model of evolutionary change in proteins”; “Atlas ofProtein Sequence and Structure,” Vol. 5, Suppl. 3 (Ed. M. O. Dayhoff),pp. 345-352, Natl. Biomed. Res. Round., Washington, D.C.; Henikoff etal. (1992) Proc. Natl. Acad. Sci. USA, 89:10915-10919. The BLOSUM62matrix is often used as a default scoring substitution matrix insequence alignment protocols such as Gapped BLAST 2.0. The gap existencepenalty is imposed for the introduction of a single amino acid gap inone of the aligned sequences, and the gap extension penalty is imposedfor each additional empty amino acid position inserted into an alreadyopened gap. The alignment is defined by the amino acids position of eachsequence at which the alignment begins and ends, and optionally by theinsertion of a gap or multiple gaps in one or both sequences so as toarrive at the highest possible score. While optimal alignment andscoring can be accomplished manually, the process is facilitated by theuse of a computer-implemented alignment algorithm, e.g., gapped BLAST2.0, described in Altschul, et al. (1997) Nucleic Acids Res.,25:3389-3402, and made available to the public at the National Centerfor Biotechnology Information Website. Optimal alignments, includingmultiple alignments can be prepared using readily available programssuch as PSI-BLAST, which is described by Altschul, et al. (1997) NucleicAcids Res., 25:3389-3402.

With respect to an amino acid sequence that is optimally aligned with areference sequence, an amino acid residue “corresponds to” the positionin the reference sequence with which the residue is paired in thealignment. The “position” is denoted by a number that sequentiallyidentifies each amino acid in the reference sequence based on itsposition relative to the N-terminus Owing to deletions, insertions,truncations, fusions, and the like that must be taken into account whendetermining an optimal alignment, in general the amino acid residuenumber in a test sequence is determined by simply counting from theN-terminal will not necessarily be the same as the number of itscorresponding position in the reference sequence. For example, in a casewhere there is a deletion in an aligned test sequence, there will be noamino acid that corresponds to a position in the reference sequence atthe site of deletion. Where there is an insertion in an alignedreference sequence, that insertion will not correspond to any amino acidposition in the reference sequence. In the case of truncations orfusions there can be stretches of amino acids in either the reference oraligned sequence that do not correspond to any amino acid in thecorresponding sequence.

In a further embodiment, the present invention provides an HHDHpolypeptide that is at least 93% identical to SEQ ID NO: 200 (i.e., 18or fewer amino acid differences as compared to SEQ ID NO: 200, whenoptimally aligned with SEQ ID NO: 200). Some of these HHDH polypeptidesare at least 95% identical to SEQ ID NO: 200, and some are at least 97,98, or 99% identical to SEQ ID NO: 200. In certain embodiments, thesepolypeptides have one or more of the following residues: T at (residue)position 2, A or P or S at position 3, V at position 4, D at position 6,either I or F at position 9, L at position 10, S at position 13, S atposition 14, K at position 15, C at position 16, T or R at position 17,either C or S or K at position 20, T at position 24, Q at position 26, Fat position 28, T at position 29, A at position 30, L at position 31, Gat position 33, R at position 34, L at position 35, N at position 36, Hat position 37, D at position 40, L at position 44, P at position 45,either P or A at position 47, N at position 52, V at position 54, R atposition 55, D at position 56, K at position 58, G or D at position 61,V at position 63, R at position 72, I at position 75, P at position 76,C at position 78, Y at position 82, either S or L at position 84, A atposition 85, E at position 91, D at position 93, Q or G at position 95,N at position 96, K at position 107, A at position 112, either T, S or Gat position 114, A at position 115, P at position 117, N at position120, E at position 121, P at position 122, R at position 126, V atposition 130, S at position 133, A or V at position 134, L, W or V atposition 136, H at position 139, I or R at position 142, S at position144, S at position 146, T at position 152, S at position 153, either Sor A at position 154, V at position 168, T at position 169, F atposition 177, V at position 178, I at position 180, G or I at position181, K at position 184, Y at position 186, L at position 194, N atposition 198, M at position 199, E at position 215, G at position 236, Vat position 237, L at position 238, T at position 240, either I or A orV at position 245, Y at position 249, V or I at position 252, and V atposition 254.

The present invention also provides HHDH polypeptides encoded by anucleic acid that hybridizes under stringent conditions oversubstantially the entire length of a nucleic acid corresponding to SEQID NO: 1, where the encoded polypeptide, when optimally aligned with SEQID NO: 2, comprises an amino acid sequence having at least one aminoacid residue selected from the group consisting of T at (residue)position 2, A or P or S at position 3, V at position 4, D at position 6,either I or F at position 9, L at position 10, S at position 13, S atposition 14, K at position 15, C at position 16, T or R at position 17,either S or K at position 20, T at position 24, Q at position 26, F atposition 28, T at position 29, A at position 30, L at position 31, G atposition 33, R at position 34, L at position 35, N at position 36, H atposition 37, D at position 40, L at position 44, P at position 45,either P or A at position 47, N at position 52, V at position 54, R atposition 55, D at position 56, G or D at position 61, V at position 63,R or Q at position 72, I at position 75, P at position 76, C at position78, Y at position 82, either S or L at position 84, A at position 85, Eat position 91, D at position 93, Q or G at position 95, N at position96, G at position 99, K at position 107, A at position 112, either T, Sor G at position 114, A at position 115, P at position 117, N atposition 120, Eat position 121, P at position 122, R at position 126, Vat position 130, S at position 133, A or V at position 134, L, W or V atposition 136, H at position 139, I or R at position 142, S at position144, S at position 146, T at position 152, S at position 153, either Sor A at position 154, V at position 168, T at position 169, F atposition 177, V at position 178, I at position 180, G or I at position181, K at position 184, Y at position 186, T at position 189, L atposition 194, N at position 198, M at position 199, E at position 215, Aat position 222, G at position 236, V at position 237, L at position238, T at position 240, either I or A or V at position 245, Y atposition 249, V or I at position 252, and V at position 254.

The present invention also provides an isolated or recombinantpolypeptide having at least 1.4 fold greater HHDH activity as comparedto wild-type HHDH having the amino acid sequence of SEQ ID NO: 2, and

wherein the polypeptide is encoded by a nucleic acid that hybridizesunder stringent conditions over substantially the entire length of anucleic acid having a sequence selected from the group consisting of SEQID NO: 3, 9, 11, 13, 15, 17, 33, 37, 43, 47, 49, 51, 65, 67, 79, 83,109, 113, 115, 117, 153, 157, 161, 163, 165, 169, 179, 161, 199, 261,263, 265, 269, 421, 439, 441, 447, 469, 475, 519, 701, 725, 729, 731,733, 735, 737, and complementary sequences thereof.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well-characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) “Laboratory Techniques inbiochemistry and Molecular Biology-Hybridization with Nucleic AcidProbes,” Part I, Chapter 2 (Elsevier, New York).

As used herein, the term “stringent hybridization wash conditions” inthe context of nucleic acid hybridization experiments, such as Southernand Northern hybridizations, are sequence dependent, and are differentunder different environmental parameters. An extensive guide to thehybridization of nucleic acids is found in Tijessen (1993) “LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes,” Part I, Chapter 2 (Elsevier, New York).

For purposes of the present invention, “highly stringent” (or “highstringency”) hybridization and wash conditions are generally selected tobe about 5° C. or less lower than the thermal melting point (T_(m)) forthe specific sequence at a defined ionic strength and pH (as notedbelow, highly stringent conditions can also be referred to incomparative terms). The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the test sequence hybridizes to aperfectly matched probe. Very stringent conditions are selected to beequal to the T_(m) for a particular probe.

The T_(m) of a nucleic acid duplex indicates the temperature at whichthe duplex is 50% denatured under the given conditions and it representsa direct measure of the stability of the nucleic acid hybrid. Thus, theT_(m) corresponds to the temperature corresponding to the midpoint intransition from helix to random coil; it depends on length, nucleotidecomposition, and ionic strength for long stretches of nucleotides.

After hybridization, unhybridized nucleic acid material can be removedby a series of washes, the stringency of which can be adjusted dependingupon the desired results. Low stringency washing conditions (e.g., usinghigher salt and lower temperature) increase sensitivity, but can producenonspecific hybridization signals and high background signals (i.e.,loses specificity). Higher stringency conditions (e.g., using lower saltand higher temperature that is closer to the hybridization temperature)lowers the background signal, typically with only the specific signalremaining (i.e., increases specificity). See Rapley, R. and Walker, J.M. Eds., “Molecular Biomethods Handbook” (Humana Press, Inc. 1998).

The T_(m) of a DNA-DNA duplex can be estimated using Equation 1 asfollows:

T _(m) (° C.)=81.5° C.+16.6 (log₁₀M)+0.41 (% G+C)−0.72 (% f)−500/n,

-   -   where M is the molarity of the monovalent cations (usually Na+),        (% G+C) is the percentage of guanosine (G) and cystosine (C)        nucleotides, (% f) is the percentage of formamide and n is the        number of nucleotide bases (i.e., length) of the hybrid. See id.

The T_(m) of an RNA-DNA duplex can be estimated by using Equation 2 asfollows:

-   -   T_(m) (° C.)=79.8° C.+18.5 (log₁₀M)+0.58 (% G+C)−11.8 (%        G+C)²−0.56 (% f)−820/n, where M is the molarity of the        monovalent cations (usually Na+), (% G+C) is the percentage of        guanosine (G) and cystosine (C) nucleotides, (% f) is the        percentage of formamide and n is the number of nucleotide bases        (i.e., length) of the hybrid. Id.

Equations 1 and 2 are typically accurate only for hybrid duplexes longerthan about 100-200 nucleotides. Id.

The Tm of nucleic acid sequences shorter than 50 nucleotides can becalculated as follows:

T _(m) (° C.)=4(G+C)+2(A+T),

where A (adenine), C, T (thymine), and G are the numbers of thecorresponding nucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formamidewith 1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see Sambrook, et al., Molecular Cloning—ALaboratory Manual” (1989) Cold Spring Harbor Laboratory (Cold SpringHarbor, N.Y.) for a description of SSC buffer). Often the highstringency wash is preceded by a low stringency wash to removebackground probe signal. An example low stringency wash is 2×SSC at 40°C. for 15 minutes.

In general, a signal to noise ratio of 2.5×-5×(or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization. Detection of at leaststringent hybridization between two sequences in the context of thepresent invention indicates relatively strong structural similarity orhomology to, e.g., the nucleic acids of the present invention providedin the sequence listings herein.

As noted, “highly stringent” conditions are selected to be about 5° C.or less lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. Target sequences that areclosely related or identical to the nucleotide sequence of interest(e.g., “probe”) can be identified under highly stringent conditions.Lower stringency conditions are appropriate for sequences that are lesscomplementary.

Stringent hybridization (as well as highly stringent, ultra-highstringency, or ultra-ultra high stringency hybridization conditions) andwash conditions can be readily determined empirically for any testnucleic acid. For example, in determining highly stringent hybridizationand wash conditions, the hybridization and wash conditions are graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents, such as formamide, in thehybridization or wash), until a selected set of criteria are met. Forexample, the stringency of hybridization and wash conditions aregradually increased until a probe corresponding to SEQ ID NO: 3, 9, 11,13, 15, 17, 33, 37, 43, 47, 49, 51, 65, 67, 79, 83, 109, 113, 115, 117,153, 157, 161, 163, 165, 169, 179, 161, 199, 261, 263, 265, 269, 421,439, 441, 447, 469, 475, 519, 701 or complementary sequence thereof,binds to a perfectly matched complementary target. A test nucleic acidis said to specifically hybridize to a probe nucleic acid when ithybridizes at least ½ as well to the probe as to the perfectly matchedcomplementary target, i.e., with a signal to noise ratio at least ½ ashigh as hybridization of the probe to the target under conditions inwhich the perfectly matched probe binds to the perfectly matchedcomplementary target.

Ultra high-stringency hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10×. Atarget nucleic acid which hybridizes to a probe under such conditions,with a signal to noise ratio of at least ½ that of the perfectly matchedcomplementary target nucleic acid is said to bind to the probe underultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the stringency of hybridization and/or washconditions of the relevant hybridization assay. For example, those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10×,20×, 50×, 100×, or 500×. A target nucleic acid which hybridizes to aprobe under such conditions, with a signal to noise ratio of at least ½that of the perfectly matched complementary target nucleic acid is saidto bind to the probe under ultra-ultra-high stringency conditions.

Specific HHDH polypeptides of the present invention include those havingan amino acid sequence corresponding to SEQ ID NOS: 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258.260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314,316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 368, 370, 372,374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400,402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428,430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456,458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484,486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512,514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540,542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568,570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596,598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624,626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652,654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680,682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708,710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736,738, 740, 742 or 744. All of these HHDH polypeptides have demonstratedactivity in the assays described in Example 5A or 5B.

Exemplary HHDH polynucleotides that encode these HHDH polypeptides areprovided herein as SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321,323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377,379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405,407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433,435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461,463, 465, 467, 469, 4712, 473, 475, 477, 479, 481, 483, 485, 487, 489,491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517,519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545,547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573,575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601,603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629,631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657,659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685,687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713,715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 739, 741, and 743respectively.

HHDH polypeptides of the present invention often have HHDH activity thatis at least 1.4 fold greater HHDH activity as compared to wild-type HHDHhaving the amino acid sequence of SEQ ID NO: 2, as measured in the assaydescribed in Example 5A. Some HHDH polypeptides of the present invention(SEQ ID NOS: 740, 742, 728, 90, 92, 94, 96 and 96) have HHDH enzymeactivity that is at least 2 fold and often at least 2.4 fold up to 100fold greater than the activity of Agrobacterium sp. HHDH (SEQ ID NO: 2);the HHDH polypeptides of SEQ ID NOS: 100, 732, 734 and 736 have HHDHenzyme activity that is from 100 to 500 fold greater than the activityof Agrobacterium sp. HHDH (SEQ ID NO: 2); and the HHDH polypeptides ofSEQ ID NOS: 726 and 730 have HHDH enzyme activity that is 500 to 1000times greater than the activity of Agrobacterium sp. HHDH (SEQ ID NO:2), the enzyme activities being measured in the assay described inExample 5A.

The present invention also provides HHDH polypeptides that are variantsof the polypeptide of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258. 260, 262, 264,266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,350, 352, 354, 356, 358, 360, 362, 364, 368, 370, 372, 374, 376, 378,380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406,408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434,436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462,464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490,492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518,520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546,548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574,576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602,604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630,632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658,660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686,688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714,716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740 or 742having a substitution, deletion, and/or insertion of one to six aminoacid residues.

Variants of the HHDH polypeptides of the present invention may begenerated using methods that are well known to those having ordinaryskill in the art. Libraries of these variants may be generated andscreened using the high throughput screen for presence of HHDH activitydescribed in Example 4A. In some instances it may be desirable toidentify halohydrin dehalogenases that exhibit activity in the presenceof cyanohydrin product inhibitor, e.g., ethyl(R)-4-cyano-3-hydroxybutyrate. A high throughput screen for identifyingsuch enzymes is provided in Example 4B.

Each of the residue changes to an HHDH polypeptide was evaluated todetermine what relationship, if any, existed between the sequence changeand the desired function (increased HHDH enzymatic activity). To do so,the sequence changes and resulting enzyme activity in members of alibrary generated by the method described in WO 00/42561 were evaluatedusing the method disclosed in U.S. Ser. No. 10/379,378 filed Mar. 3,2003, entitled “Methods, systems, and software for identifyingfunctional biomolecules” and incorporated herein by reference. Basedupon this method, codons encoding important residues at certainpositions that appear to correlate favorably to activity were identifiedand incorporated into the polynucleotides of a subsequently generatedcombinatorial library. In other words, the polynucleotides encoding thedesired change were generated, expressed and then screened. The methodis again applied to the resulting sequences and the enzymatic activityof the hits. The results are again utilized to select those residuechanges that enhance enzyme activity for programming into the nextlibrary. Using this method, the functionality of various sequencechanges (and although not characterized, potential structural changes aswell) is subject to immediate evaluation. The residue changes at variousresidue positions that provide for enhanced enzymatic activity relativeto the wild-type HHDH are disclosed herein in the sequences andelsewhere as preferred residues at identified positions.

Those variants exhibiting the presence of HHDH activity can be furthercharacterized in the quantitative HHDH assay described in Example 5A.Variants that exhibit HHDH activity in the presence of productcyanohydrin, e.g., ethyl (R) 4-cyano-3-hydroxybutyrate, may be furthercharacterized using the assay described in Example 5B. Example 5Bdescribes a protocol for assaying for enzymes that are robust withrespect to product inhibition. Thus, variant libraries may be readilyscreened and assayed to identify HHDH polypeptides that are active underconditions that mimic actual process conditions. The present inventionprovides HHDH polypeptides that exhibit significant activity even in thepresence of product, ethyl (R)-4-cyano-3-hydroxybutyrate in the assaydescribed in Example 5B (e.g., SEQ ID NOS: 98, 100, 102, 104, 106, 108,120, 122, 124, 126, 128, 130, 132, 136, 138, 140, 142, 144, 146, 148,150, 152, 160, 174, 176, 178, 188, 190, 192, 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258,260, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 300, 302,304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330,332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358,360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386,388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414,416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442,444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470,472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498,500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526,528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554,556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582,584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610,612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638,640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666,668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694,696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722,724, 726, 728, 730, 732, 734, 736, 738, 740 or 742. Polypeptides thatexhibit the ability to convert ethyl (S)-4-chloro-3-hydroxybutyrate toethyl (R)-4-cyano-3-hydroxybutyrate in the assay of Example 5B, wouldalso demonstrate HHDH activity in the assay of Example 5A.

Methods for generating variant libraries are well known in the art. Forexample, mutagenesis and directed evolution methods can be readilyapplied to polynucleotides (such as, for example, wild-type HHDHencoding polynucleotides or the polynucleotides of the presentinvention) to generate variant libraries that can be expressed,screened, and assayed using the methods described herein. Mutagenesisand directed evolution methods are well known in the art. See, e.g.,Ling, et al., “Approaches to DNA mutagenesis: an overview,” Anal.Biochem., 254(2):157-78 (1997); Dale, et al., “Oligonucleotide-directedrandom mutagenesis using the phosphorothioate method,” Methods Mol.Biol., 57:369-74 (1996); Smith, “In vitro mutagenesis,” Ann. Rev.Genet., 19:423-462 (1985); Botstein, et al., “Strategies andapplications of in vitro mutagenesis,” Science, 229:1193-1201 (1985);Carter, “Site-directed mutagenesis,” Biochem. J., 237:1-7 (1986);Kramer, et al., “Point Mismatch Repair,” Cell, 38:879-887 (1984); Wells,et al., “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites,” Gene, 34:315-323 (1985); Minshull,et al., “Protein evolution by molecular breeding,” Current Opinion inChemical Biology, 3:284-290 (1999); Christians, et al., “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling,” Nature Biotechnology, 17:259-264 (1999); Crameri, et al.,“DNA shuffling of a family of genes from diverse species acceleratesdirected evolution,” Nature, 391:288-291; Crameri, et al., “Molecularevolution of an arsenate detoxification pathway by DNA shuffling,”Nature Biotechnology, 15:436-438 (1997); Zhang, et al., “Directedevolution of an effective fucosidase from a galactosidase by DNAshuffling and screening,” Proceedings of the National Academy ofSciences, U.S.A., 94:45-4-4509; Crameri, et al., “Improved greenfluorescent protein by molecular evolution using DNA shuffling,” NatureBiotechnology, 14:315-319 (1996); Stemmer, “Rapid evolution of a proteinin vitro by DNA shuffling,” Nature, 370:389-391 (1994); Stemmer, “DNAshuffling by random fragmentation and reassembly: In vitro recombinationfor molecular evolution,” Proceedings of the National Academy ofSciences, U.S.A., 91:10747-10751 (1994); WO 95/22625; WO 97/0078; WO97/35966; WO 98/27230; WO 00/42651; and WO 01/75767.

In another embodiment, the present invention also provides a fragment ofthe HHDH polypeptides described herein having HHDH activity that is atleast 1.4 fold greater than the activity of Agrobacterium sp.(wild-type) HHDH (SEQ ID NO: 2) in the assay of Example 5A. As usedherein, the term “fragment” refers to a polypeptide having a deletion offrom 1 to 5 amino acid residues from the carboxy terminus, the aminoterminus, or both. Preferably, the deletion is from 1 to 5 residues fromthe carboxy terminus

HHDH Polynucleotides

The present invention provides polynucleotides that encode HHDHpolypeptides of the present invention. In a specific embodiment of thepresent invention, HHDH polynucleotides comprise a nucleic acid thathybridizes under stringent conditions over substantially the entirelength of a nucleic acid corresponding to SEQ ID NO: 1, where thepolypeptide encoded by the HHDH polynucleotide comprises an amino acidsequence having at least one amino acid residue selected from the groupconsisting of T at (residue) position 2, A or P or S at position 3, V atposition 4, D at position 6, either I or F at position 9, L at position10, S at position 13, S at position 14, K at position 15, C at position16, T or R at position 17, either C or S or K at position 20, T atposition 24, Q at position 26, F at position 28, T at position 29, A atposition 30, L at position 31, G at position 33, R at position 34, L atposition 35, N at position 36, H at position 37, D at position 40, L atposition 44, P at position 45, either P or A at position 47, N atposition 52, V at position 54, R at position 55, D at position 56, K atposition 58, G or D at position 61, V at position 63, R at position 72,I at position 75, P at position 76, C at position 78, Y at position 82,either S or L at position 84, A at position 85, Eat position 91, D atposition 93, Q or G at position 95, N at position 96, K at position 107,A at position 112, either T, S or G at position 114, A at position 115,P at position 117, N at position 120, E at position 121, P at position122, R at position 126, V at position 130, S at position 133, A or V atposition 134, L, W or V at position 136, H at position 139, I or R atposition 142, S at position 144, S at position 146, T at position 152, Sat position 153, either S or A at position 154, V at position 168, T atposition 169, F at position 177, V at position 178, I at position 180, Gor I at position 181, K at position 184, Y at position 186, L atposition 194, N at position 198, M at position 199, E at position 215, Gat position 236, V at position 237, L at position 238, T at position240, either I or A or V at position 245, Y at position 249, V or I atposition 252, and V at position 254, when optimally aligned with SEQ IDNO: 2. The present invention also provides an HHDH polynucleotide, SEQID NO: 1, that is codon optimized for expression in E. coli. Thepolypeptide encoded by this codon optimized polynucleotide correspondsto HHDH polypeptide from Agrobacterium sp. (SEQ ID NO: 2).

In addition, the present invention provides specific polynucleotidescorresponding to SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295,297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323,325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351,353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379,381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407,409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435,437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463,465, 467, 469, 4712, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491,493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519,521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547,549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575,577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603,605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631,633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659,661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687,689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715,717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 739, and 741.

Those having ordinary skill in the art will readily appreciate that dueto the degeneracy of the genetic code, a multitude of nucleotidesequences encoding HHDH polypeptides of the present invention exist.Table I is a Codon Table that provides the synonymous codons for eachamino acid. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU allencode the amino acid arginine. Thus, at every position in the nucleicacids of the invention where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons described abovewithout altering the encoded polypeptide. It is understood that U in anRNA sequence corresponds to T in a DNA sequence.

TABLE 1 Codon Table Amino acids Codon Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

Such “silent variations” are one species of “conservative” variation.One of ordinary skill in the art will recognize that each codon in anucleic acid (except AUG, which is ordinarily the only codon formethionine) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention contemplates and provides each andevery possible variation of nucleic acid sequence encoding a polypeptideof the invention that could be made by selecting combinations based onpossible codon choices. These combinations are made in accordance withthe standard triplet genetic code (set forth in Table 1), as applied tothe polynucleotide sequences of the present invention.

A group of two or more different codons that, when translated in thesame context, all encode the same amino acid, are referred to herein as“synonymous codons.” HHDH polynucleotides of the present invention maybe codon optimized for expression in a particular host organism bymodifying the polynucleotides to conform with the optimum codon usage ofthe desired host organism. Those having ordinary skill in the art willrecognize that tables and other references providing preferenceinformation for a wide range of organisms are readily available Seee.g., Henaut and Danchin in “Escherichia coli and Salmonella,”Neidhardt, et al. Eds., ASM Press, Washington D.C. (1996), pp.2047-2066.

An exemplary HHDH variant polynucleotide sequence of the presentinvention is provided as SEQ ID NO: 31, which expresses well in E. coli.This polynucleotide is a variant of SEQ ID NO: 1 that expresses thepolypeptide corresponding to SEQ ID NO: 2 from E. coli at a level ofabout 4½ fold higher than the amount expressed from SEQ ID NO: 1 (i.e.,HHDH-encoding polynucleotide encoding native HHDH from Agrobacteriumsp.).

In some embodiments of the present invention, certain codons arepreferred when the following residues are employed in the HHDHpolypeptides of the present invention: ATT encoding Isoleucine at aminoacid position 5; AAG encoding Lysine at amino acid position 36; ATTencoding Isoleucine at amino acid position 63; GAG encoding Glutamicacid at amino acid position 95; and CCC encoding Proline at amino acidposition 188. The amino acid position referred to above is thecorresponding amino acid position in SEQ ID NO: 2, when the inventionHHDH polypeptides are aligned with SEQ ID NO: 2.

The terms “conservatively modified variations” and “conservativevariations” are used interchangeably herein to refer to those nucleicacids that encode identical or essentially identical amino acidsequences, or in the situation where the nucleic acids are not codingsequences, the term refers to nucleic acids that are identical. One ofordinary skill in the art will recognize that individual substitutions,deletions or additions which alter, add or delete a single amino acid ora small percentage of amino acids in an encoded sequence are consideredconservatively modified variations where the alterations result in oneor more of the following: the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. When more than one amino acid is affected, the percentage istypically less than 5% of amino acid residues over the length of theencoded sequence, and more typically less than 2%. References providingamino acids that are considered conservative substitutions for oneanother are well known in the art.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagines), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine, proline, cysteine andmethionine). Amino acid substitutions which do not generally alter thespecific activity are known in the art and are described, for example,by H. Neurath and R. L. Hill, 1979, in “The Proteins,” Academic Press,New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly aswell as these in reverse.

Conservatively substituted variations of the HHDH polypeptides of thepresent invention include substitutions of a small percentage, typicallyless than 5%, more typically less than 2%, and often less than 1% of theamino acids of the polypeptide sequence, with a conservatively selectedamino acid of the same conservative substitution group. The addition ofsequences which do not alter the encoded activity of an HHDHpolynucleotide, such as the addition of a non-functional or non-codingsequence, is considered a conservative variation of the HHDHpolynucleotide.

Polynucleotides of the present invention can be prepared using methodsthat are well known in the art. Typically, oligonucleotides of up toabout 120 bases are individually synthesized, then joined (e.g., byenzymatic or chemical ligation methods, or polymerase-mediated methods)to form essentially any desired continuous sequence. For example,polynucleotides of the present invention can be prepared by chemicalsynthesis using, e.g., the classical phosphoramidite method described byBeaucage, et al. (1981) Tetrahedron Letters, 22:1859-69, or the methoddescribed by Matthes, et al. (1984) EMBO J., 3:801-05., e.g., as istypically practiced in automated synthetic methods. According to thephosphoramidite method, oligonucleotides are synthesized, e.g., in anautomatic DNA synthesizer, purified, annealed, ligated and cloned inappropriate vectors.

In addition, essentially any nucleic acid can be custom ordered from anyof a variety of commercial sources, such as The Midland CertifiedReagent Company (Midland, Tex.), The Great American Gene Company(Ramona, Calif.), ExpressGen Inc. (Chicago, Ill.), Operon TechnologiesInc. (Alameda, Calif.), and many others.

Polynucleotides may also be synthesized by well-known techniques asdescribed in the technical literature. See, e.g., Carruthers, et al.,Cold Spring Harbor Symp. Quant. Biol., 47:411-418 (1982) and Adams, etal., J. Am. Chem. Soc., 105:661 (1983). Double stranded DNA fragmentsmay then be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

General texts which describe molecular biological techniques usefulherein, including the use of vectors, promoters and many other relevanttopics, include Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif. (Berger); Sambrook et al., Molecular Cloning—A LaboratoryManual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989 (“Sambrook”) and Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(supplemented through 1999) (“Ausubel”). Examples of protocolssufficient to direct persons of skill through in vitro amplificationmethods, including the polymerase chain reaction (PCR) the ligase chainreaction (LCR), Qβ-replicase amplification and other RNA polymerasemediated techniques (e.g., NASBA), e.g., for the production of thehomologous nucleic acids of the invention are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94;(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.Clin. Chem. 35, 1826; Landegren et al., (1988) Science 241, 1077-1080;Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene4, 560; Barringer et al. (1990) Gene 89, 117, and Sooknanan and Malek(1995) Biotechnology 13: 563-564. Improved methods for cloning in vitroamplified nucleic acids are described in Wallace et al., U.S. Pat. No.5,426,039. Improved methods for amplifying large nucleic acids by PCRare summarized in Cheng et al. (1994) Nature 369: 684-685 and thereferences cited therein, in which PCR amplicons of up to 40 kb aregenerated. One of ordinary skill in the art will readily appreciate thatessentially any RNA can be converted into a double stranded DNA suitablefor restriction digestion, PCR expansion and sequencing using reversetranscriptase and a polymerase. See, e.g., Ausubel, Sambrook and Berger,all supra.

Vectors, Promoters, and Expression Systems

The present invention also includes recombinant constructs comprisingone or more of the HHDH polynucleotide sequences as broadly describedabove. The term “construct” or “nucleic acid construct” refers herein toa nucleic acid, either single- or double-stranded, which is isolatedfrom a naturally occurring gene or which has been modified to containsegments of nucleic acids in a manner that would not otherwise exist innature. The term “nucleic acid construct” is synonymous with the term“expression cassette” when the nucleic acid construct contains thecontrol sequences required for expression of an HHDH coding sequence ofthe present invention.

The present invention also provides an expression vector comprising anHHDH polynucleotide of the present invention operably linked to apromoter. Example 1 provides a description of how to make expressionconstructs for expression of halohydrin dehalogenase. The term “controlsequences” refers herein to all the components that are necessary oradvantageous for the expression of a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

The term “operably linked” refers herein to a configuration in which acontrol sequence is appropriately placed at a position relative to thecoding sequence of the DNA sequence such that the control sequencedirects the expression of a polypeptide.

When used herein, the term “coding sequence” is intended to cover anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon. The coding sequence typically includes a DNA, cDNA, and/orrecombinant nucleotide sequence.

As used herein, the term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

The term “expression vector” refers herein to a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of theinvention, and which is operably linked to additional segments thatprovide for its transcription.

As used herein, the term “host cell” refers to any cell type which issusceptible to transformation with a nucleic acid construct.

Nucleic acid constructs of the present invention comprise a vector, suchas, a plasmid, a cosmid, a phage, a virus, a bacterial artificialchromosome (BAC), a yeast artificial chromosome (YAC), or the like, intowhich a nucleic acid sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available.

Polynucleotides of the present invention can be incorporated into anyone of a variety of expression vectors suitable for expressing apolypeptide. Suitable vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids;phage DNA; baculovirus; yeast plasmids; vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associatedvirus, retroviruses and many others. Any vector that transduces geneticmaterial into a cell, and, if replication is desired, which isreplicable and viable in the relevant host can be used.

When incorporated into an expression vector, a polynucleotide of theinvention is operatively linked to an appropriate transcription controlsequence (promoter) to direct mRNA synthesis, e.g., T5 promoter.Examples of such transcription control sequences particularly suited foruse in transgenic plants include the cauliflower mosaic virus (CaMV) andfigwort mosaic virus (FMV). Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses and whichcan be used in some embodiments of the invention include SV40 promoter,E. coli lac or trp promoter, phage lambda P_(L) promoter, tac promoter,T7 promoter, and the like. An expression vector optionally contains aribosome binding site for translation initiation, and a transcriptionterminator, such as PinII. The vector also optionally includesappropriate sequences for amplifying expression, e.g., an enhancer.

In addition, the expression vectors of the present invention optionallycontain one or more selectable marker genes to provide a phenotypictrait for selection of transformed host cells. Suitable marker genesinclude those coding for resistance to the antibiotic spectinomycin orstreptomycin (e.g., the aada gene), the streptomycin phosphotransferase(SPT) gene coding for streptomycin resistance, the neomycinphosphotransferase (NPTII) gene encoding kanamycin or geneticinresistance, the hygromycin phosphotransferase (HPT) gene coding forhygromycin resistance. Additional selectable marker genes includedihydrofolate reductase or neomycin resistance for eukaryotic cellculture, and tetracycline or ampicillin resistance in E. coli.

An exemplary expression vector for the expression of HHDH polypeptidesof the present invention is depicted in FIG. 1. Vectors of the presentinvention can be employed to transform an appropriate host to permit thehost to express an invention protein or polypeptide. Examples ofappropriate expression hosts include bacterial cells, such as E. coli,B. subtilis, and Streptomyces. In bacterial systems, a number ofexpression vectors may be selected, such as, for example,multifunctional E. coli cloning and expression vectors.

HHDH polynucleotides of the invention can also be fused, for example,in-frame to nucleic acids encoding a secretion/localization sequence, totarget polypeptide expression to a desired cellular compartment,membrane, or organelle of a cell, or to direct polypeptide secretion tothe periplasmic space or into the cell culture media. Such sequences areknown to those of skill, and include secretion leader peptides,organelle targeting sequences (e.g., nuclear localization sequences,endoplasmic reticulum (ER) retention signals, mitochondrial transitsequences, chloroplast transit sequences), membrane localization/anchorsequences (e.g., stop transfer sequences, GPI anchor sequences), and thelike.

Expression Hosts

The present invention also relates to engineered host cells that aretransduced (transformed or transfected) with a vector or construct ofthe invention (e.g., an invention cloning vector or an inventionexpression vector), as well as the production of polypeptides of theinvention by recombinant techniques. The vector may be, for example, aplasmid, a viral particle, a phage, etc. The host cell can be aeukaryotic cell, such as a plant cell. Alternatively, the host cell canbe a prokaryotic cell, such as a plant cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, electroporation, orother common techniques (Davis, L., Dibner, M. and Battey, I. (1986)Basic Methods in Molecular Biology). The engineered host cells can becultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transformants, or amplifying the HHDHpolynucleotide. Culture conditions, such as temperature, pH and thelike, are those previously used with the host cell selected forexpression, and will be apparent to those skilled in the art and in thereferences cited herein, including, e.g., Sambrook, Ausubel and Berger,as well as e.g., Freshney (1994) Culture of Animal Cells, a Manual ofBasic Technique, third edition, Wiley-Liss, New York and the referencescited therein.

HHDH polypeptides of the invention can be produced in non-animal cellssuch as plants, yeast, fungi, bacteria, and the like. In addition toSambrook, Berger and Ausubel, details regarding non-animal cell culturecan be found in Payne et al. (1992) Plant Cell and Tissue Culture inLiquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg andPhillips (eds) (1995) Plant Cell, Tissue and Organ Culture; FundamentalMethods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg NewYork) and Atlas and Parks (eds) The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, Fla. The host cell can be a eukaryoticcell, such as a plant cell. Alternatively, the host cell can be aprokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, electroporation, orother common techniques (Davis, L., Dibner, M., and Battey, I. (1986)Basic Methods in Molecular Biology).

Fusion Polypeptides for Purification

HHDH polypeptides of the present invention may also be expressed as partof a fusion polypeptide to facilitate purification of the encoded HHDHpolypeptide. Polynucleotides encoding such fusion polypeptides comprisea nucleic acid sequence corresponding to an HHDH polynucleotide of thepresent invention that is fused-in frame to a purification facilitatingdomain. As used herein, the term “purification facilitating domain”refers to a domain that mediates purification of the polypeptide towhich it is fused. Suitable purification domains include metal chelatingpeptides, histidine-tryptophan modules that allow purification onimmobilized metals, a sequence which binds glutathione (e.g., GST), ahemagglutinin (HA) tag (corresponding to an epitope derived from theinfluenza hemagglutinin protein; Wilson et al. (1984) Cell, 37:767),maltose binding protein sequences, the FLAG epitope utilized in theFLAGS extension/affinity purification system (Immunex Corp, Seattle,Wash.), and the like. The inclusion of a protease-cleavable polypeptidelinker sequence between the purification domain and the HHDH polypeptideis useful to facilitate purification. One expression vector contemplatedfor use in the compositions and methods described herein provides forexpression of a fusion protein comprising a polypeptide of the inventionfused to a polyhistidine region separated by an enterokinase cleavagesite. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography, as described in Porathet al. (1992) Protein Expression and Purification 3:263-281) while theenterokinase cleavage site provides a means for separating the HHDHpolypeptide from the fusion protein. pGEX vectors (Promega; Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to ligand-agarose beads (e.g., glutathione-agarose in thecase of GST-fusions) followed by elution in the presence of free ligand.

Production and Recovery of HHDH Polypeptides

The present invention further provides a method of making an HHDHpolypeptide, said method comprising: (a) cultivating a host celltransformed with an HHDH polynucleotide under conditions suitable forthe production of the HHDH polypeptide; and (b) recovering the HHDHpolypeptide. Typically, recovery is from the host cell culture medium,the host cell or both, using protein recovery techniques that are wellknown in the art, including those described below.

Following transduction of a suitable host strain and growth(cultivating) of the host strain to an appropriate cell density, theselected promoter is induced by appropriate means (e.g., temperatureshift or chemical induction) and cells are cultured for an additionalperiod. Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification. Microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents, or other methods, which are well known to those skilled in theart.

As noted, many references are available for the culture and productionof many cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin. See e.g., Sambrook, Ausubel, andBerger (all supra), as well as Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Doyle and Griffiths (1997) Mammalian CellCulture: Essential Techniques John Wiley and Sons, NY; Humason (1979)Animal Tissue Techniques, fourth edition W.H. Freeman and Company; andRicciardelli, et al., (1989) In vitro Cell Dev. Biol. 25:1016-1024. Forplant cell culture and regeneration, Payne et al. (1992) Plant Cell andTissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.;Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture;Fundamental Methods Springer Lab Manual, Springer-Verlag (BerlinHeidelberg New York); Jones, ed. (1984) Plant Gene Transfer andExpression Protocols, Humana Press, Totowa, N.J. and Plant MolecularBiology (1993) R. R. D. Croy, Ed. Bios Scientific Publishers, Oxford,U.K. ISBN 0 12 198370 6. Cell culture media in general are set forth inAtlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRCPress, Boca Raton, Fla. Additional information for cell culture is foundin available commercial literature such as the Life Science ResearchCell Culture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.)(“Sigma-LSRCCC”) and, e.g., The Plant Culture Catalogue and supplement(1997) also from Sigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-PCCS”).

HHDH polypeptides of the present invention can be recovered and purifiedfrom recombinant cell cultures by any of a number of methods well knownin the art, including ammonium sulfate or solvent (e.g., ethanol,acetone, and the like) precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography (e.g., using any ofthe tagging systems noted herein), hydroxylapatite chromatography, andlectin chromatography. Protein refolding steps can be used, as desired,in completing the configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed in the finalpurification steps. In addition to the references noted supra, a varietyof purification methods are well known in the art, including, e.g.,those set forth in Sandana (1997) Bioseparation of Proteins, AcademicPress, Inc.; and Bollag et al. (1996) Protein Methods, 2^(nd) Edition,Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook HumanaPress, NJ, Harris and Angal (1990) Protein Purification Applications: APractical Approach, IRL Press at Oxford, Oxford, England; Harris andAngal Protein Purification Methods: A Practical Approach, IRL Press atOxford, Oxford, England; Scopes (1993) Protein Purification: Principlesand Practice 3^(rd) Edition, Springer Verlag, NY; Janson and Ryden(1998) Protein Purification: Principles, High Resolution Methods andApplications, Second Edition, Wiley-VCH, NY; and Walker (1998) ProteinProtocols on CD-ROM, Humana Press, NJ.

In some cases it may be desirable to produce the HHDH polypeptides ofthe invention on a large scale suitable for industrial and/or commercialapplications. In such cases bulk fermentation procedures are employed.An exemplary bulk fermentation procedure for producing HHDH is providedin Example 2. Briefly, an HHDH polynucleotide, is cloned into anexpression vector, such as, for example, the vector depicted in FIG. 1(PCK110700). After inserting the polynucleotide of interest into avector, the vector is transformed into a bacterial host, such as, forexample, E. coli BL21 (Strategene, La Jolla, Calif.) after passagethrough E. coli TOP 10 (Invitrogen, Carlsbad, Calif.) using standardmethods.

The transformed cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods that are known in the art.For example, the cell may be cultivated by shake flask cultivation,small-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). The secreted polypeptide can be recovereddirectly from the nutrient (culture) medium.

The resulting polypeptide may be isolated by methods known in the art.For example, the polypeptide may be isolated from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray-drying, evaporation, or precipitation. Theisolated polypeptide may then be further purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), or extraction (see, e.g., Bollag et al.(1996) Protein Methods, 2^(nd) Edition, Wiley-Liss, NY; Walker (1996)The Protein Protocols Handbook, Humana Press, NJ; Bollag et al. (1996)Protein Methods, 2^(nd) Edition, Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook, Humana Press, NJ). A procedure forrecovering the HHDH polypeptide from a cell lysate is illustrated inExample 3.

It is believed that the pI of the wild-type HHDH of SEQ ID NO: 2 maybetoo low for polyethyleneimine (PEI) precipitation to be used to purifyHHDH from DNA. Applicants have discovered that they could make thefollowing residue changes relative to the alignment in SEQ ID NO: 2 toproduce HHDH polypeptides of the present invention that have asufficiently high pI to allow for isolation by PEI precipitation, butwithout loss of HHDH enzyme activity: E40Q,K, E42Q,K, E46Q,K, E56Q,K,E58Q,K, E61Q,K, and E64Q,K. Thus, in another embodiment, the presentinvention is directed to an HHDH polypeptide that can be isolated fromsolution by PEI precipitation, the HHDH polypeptide, when aligned withSEQ ID NO: 2, having five or more of the residue changes selected fromthe group consisting of E40Q,K, E42Q,K, E46Q,K, E56Q,K, E58Q,K, E61Q,K,and E64Q,K. For example, PEI precipitation was applied to the HHDHpolypeptide of SEQ ID NO: 744:

MSTAIVTNVKHFGGMGSALRLSEAGHTVACHDESFKHQDQLKAFAKTYPQLIPMSEQEPAELIEAVTSALGQVDVLVSNDIYPVEWRPIDKYAVEDYRGTVEALQIKPFALVNAVASQMKKRKSGHIIFITSAAPFGPWKELSTYSSARAGASALANALSKELGEYNIPVFAIAPNYLHSGDSPYYYPTEPWKTSPEHVAHVRKVTALQRLGTQKELGELVAFLASGSCDYLTGQVFWLTGGFPVIERWPGMPE.This polypeptide is encoded by the polynucleotide of SEQ ID NO: 743:

atgagcaccgctattgtcaccaacgtcaaacattttggaggtatgggtagcgctctgcgtctgagcgaagctggtcataccgtcgcttgccatgatgaaagctttaagcatcaggatcaactgaaagcttttgctaaaacctacccacagctgatcccaatgagcgaacaggaaccagctgaactgattgaagctgtcaccagcgctcttggtcaggtcgatgtactggtcagcaacgatatctatcctgtggaatggcggccaatcgataaatacgctgtcgaggattacaggggtactgtcgaagctctgcagatcaagccatttgctctagtgaatgctgtcgcttcgcaaatgaagaagcgaaagtcggggcacatcatcttcatcacttcggctgccccgttcgggccatggaaggagctatcgacttactcttcggctcgagctggggctagtgcactagctaatgctctatcgaaggagctaggagagtacaatatcccggtgttcgctatcgctccgaattacctacactcgggggattcgccgtactattaccccactgagccgtggaagacttctccggagcacgtggctcacgtgcgtaaggtgactgctctacaacgactagggactcaaaaagagctgggggaattggtggcatttttggcatctggctcttgtgattatttgactggccaggtgttttggttgacaggcggctttcccgtcatcgaacgttggcccggcatgcccgaataatgaggatccggccaaactgttgtccgtctgcatcacctctaggtaatgtga gcggatacgatgccc.

Cell-free transcription/translation systems can also be employed toproduce HHDH polypeptides using the polynucleotides of the presentinvention. Several such systems are commercially available. A generalguide to in vitro transcription and translation protocols is found inTymms (1995) In vitro Transcription and Translation Protocols: Methodsin Molecular Biology, Volume 37, Garland Publishing, NY.

Ethyl-4-chloroacetoacetate (ECAA) is the substrate for the coupledreduction reaction using KRED/GDH to produce ethyl(S)-4-chloro-3-hydroxybutyrate (ECHB). The ECHB is then used assubstrate for the HHDH reaction. However, the ECAA starting material isa potent inhibitor (K_(i) approximately=70 μM) of HHDH. Because theKRED/GDH catalyzed reaction may go to 99.9% completion, instead of thedesired 99.97%, then 0.1% ECAA remains in the ECHB material and this0.1% ECAA can inhibit the HHDH reaction. In other words, the remainingsubstrate from the first reaction is an inhibitor in the secondreaction. Hence, it is desirable that the HHDH polypeptides of thepresent invention have resistance to inhibition by ECAA.

Applicants have discovered that they could make the following residuechanges relative to the alignment in SEQ ID NO: 2 to produce HHDHpolypeptides of the present invention that demonstrate increasedresistance against inhibition by ECAA: A4V, A82Y, A134V, G136W, G136V,L142R, L178V, W238L, A240T, W249Y, M252I. Thus, in another embodiment,the present invention is directed to an HHDH polypeptide is resistant toinhibition by ECAA, the HHDH polypeptide, when aligned with SEQ ID NO:2, having one or more of the residue changes selected from the groupconsisting of A4V, F82Y, T134V, F136W, F136V, L142R, L178V, W238L,A240T, W249Y and M252I.

A method for testing the HHDH polypeptides of the present invention fortheir reactivity in the presence of ECAA is disclosed in Example 5Cherein. A gas chromatographic method for screening the reaction productsfrom Example 5C, and determining the amount of product produced, isdisclosed in Example 6B herein.

Methods of Using HHDH Polypeptides

As described supra, HHDH polypeptides of the present invention can beused to catalyze the conversion of 4-halo-3-hydroxybutyric acidderivatives to 4-nucleophile substituted-3-hydroxybutyric acidderivatives. The novel halohydrin dehalogenases of the present inventionare also useful in the process for enzymatically resolving a mixture ofenantiomeric epoxides by reacting the mixture with an anionicnucleophile in the presence of the halohydrin dehalogenase, wherein theenzyme preferentially reacts one of the epoxide enantiomers with thenucleophile to form a mixture of the resulting enantiomerically enrichedvicinal nucleophile-substituted alcohol and the unreacted epoxideenriched in the other enantiomer, in the manner disclosed in publicationWO 01/90397, which is incorporated herein by reference in its entirety.

The foregoing and other aspects of the invention may be betterunderstood in connection with the following non-limiting examples.

EXAMPLES Example 1 Construction of Expression Constructs for Expressionof Halohydrin Dehalogenase

The gene for Agrobacterium sp. halohydrin dehalogenase was codonoptimized (SEQ ID NO: 1) for expression in E. coli based on the aminoacid sequence of the halohydrin dehalogenase from Agrobacterium sp. (SEQID NO: 2). The gene was synthesized using 60-mer oligomers, and clonedinto expression vector PCK110700 (depicted in FIG. 1) under the controlof a T5 promoter. The vector was transformed into E. coli TOP10(Invitrogen, Carlsbad, Calif.) from which plasmid DNA was prepared usingstandard methods. The plasmid DNA was then transformed into E. coli BL21(Stratagene, La Jolla, Calif.), the expression host, using standardmethods. A clone was found in the expression library that expressedactive HHDH. The gene from this clone was sequenced (see SEQ ID NO: 1(HHDH.1)) and found to encode Agrobacterium sp. HHDH (SEQ ID NO: 2).

Polynucleotides encoding halohydrin dehalogenases of the presentinvention were similarly cloned into vector PCK 110700, depicted in FIG.1, then transformed and expressed from E. coli BL21 after passagethrough E. coli TOP 10 using standard methods.

Example 2 Production of HHDH

In an aerated agitated fermentor, 10.0 L of growth medium containing0.528 g/L ammonium sulphate; 7.5 g/L of di-potassium hydrogen phosphatetrihydrate; 3.7 g/L of potassium dihydrogen phosphate; 2 g/L ofTastone-154 yeast extract; 0.05 g/L ferrous sulphate; and 3 ml/L of atrace element solution containing 2 g/L of calcium chloride dihydrate,2.2 g/L of zinc sulfate septahydrate, 0.5 g/L manganese sulfatemonohydrate, 1 g/L cuprous sulfate heptahydrate: 0.1 g/l sodium boratedecahydrate and 0.5 g/L EDTA, was brought to a temperature of 30° C. Thefermentor was inoculated with a late exponential culture of Escherchiacoli BL21 (Stratagene, La Jolla, Calif.) equipped with plasmidcontaining HHDH polynucleotides as described in Example 1, then grown ina shake flask containing LB, 1% glucose (Sigma Chemical Co., St. Louis,Mo.), and 30 μg/ml chloroamphenicol (Sigma Chemical Co., St. Louis, Mo.)to a starting optical density at 600 nm (OD₆₀₀) of 0.5 to 2.0. Thefermentor was agitated at 500-1500 rpm and air was supplied to thefermentation vessel at 1.0-15.0 L/min to maintain a dissolved oxygenlevel of 30% saturation or greater. The pH of the culture was controlledat 7.0 by addition of 20% v/v ammonium hydroxide. After the culturereached an OD₆₀₀ of 40, the temperature was maintained at 30° C. and theexpression of halohydrin dehalogenase was induced by the addition ofisopropyl-β-D-thiogalactoside (IPTG) (Sigma Chemical Corp., St. Louis,Mo.) to a final concentration of 1 mM. The culture was grown for another15 hours. After the induction, the cells were harvested bycentrifugation and washed with 10 mM potassium phosphate buffer, pH 7.0.The cell paste was used directly in the downstream recovery process orwas stored at −80° C. until use.

Example 3 Enzyme Preparation

The cell paste from Example 2 was washed by suspending 1 volume wetweight of cell paste in 3 volumes of 100 mM Tris/sulfate (pH 7.2)followed by centrifugation at 5000 g for 40 minutes in a Sorval 12BP.The washed cell paste was suspended in 2 volumes of 100 mM Tris/sulfate(pH 7.2). The intracellular HHDH was released from the cells by passingthe suspension through a homogenizer in two passes using a pressure of14,000 psig for the first pass and 8,000 psig for the second pass. Thecell lysate was allowed to cool to 4° C. between passes through thehomogenizer. The lysate is warmed to room temperature then either 2.5MMnSO₄ (50-350 mM final concentration), or a 10% w/v solution ofpolyethyleneimine (PEI), pH 7.2, (0.6-1.0% w/v final concentration) wasadded to the lysate and stirred for 30 minutes. The homogenate wascentrifuged at between 5,000 and 10,000 g in a standard laboratorycentrifuge for 30 to 60 minutes. The supernatant was desalted,concentrated by ultrafiltration, dispensed in shallow containers, frozenat −20° C. and lyophilized to a powder that was stored at −80° C.

To assess the quality of the preparation after fermentation, cell lysatecontaining the expressed halohydrin dehalogenase enzyme was assayedaccording to the following protocol. Approximately 50 μl of clarifiedcell lysate in 100 mM Tris-SO₄, 100 mM NaCN, pH 8.0 was mixed with 10 mMethyl-(S)-4-chloro-3-hydroxybutyrate (ECHB) (Sigma Aldrich, St. Louis,Mo.). The total reaction volume was 0.2 ml. The reaction was incubatedat room temperature for 30 min to 1 hour. The reaction was extractedwith 7 volumes of ethyl acetate and the organic layer removed to a 1.8ml gas chromatography (GC) vial. The organic layer was analyzed by GCfor presence of the ethyl-(R)-4-cyano-3-hydroxybutyrate product. Theamount of product produced was determined by comparison to a standardcurve prepared and analyzed under the same conditions.

Example 4 High Throughput Screen for Presence of HHDH Activity A. NoCyanohydrin in Agarose

The following screen was used to ascertain the presence of HHDHactivity. On day 1, freshly transformed colonies on a Q-tray (GenetixUSA, Inc. Beaverton, Oreg.) containing 200 ml LB agar+1% glucose, 30μg/ml chloramphenicol were picked using a Q-bot® robot colony picker(Genetix USA, Inc., Beaverton, Oreg.) into shallow 384 well Nunc platescontaining media (70 μL/well 2×YT+1% glucose, 30 μg/ml cam) (Nalge NuncInternational, Rochester, N.Y.) for overnight growth at 30° C., 250revolutions per minute (rpm), 85% relative humidity (RH). A negativecontrol (E. coli BL21 with empty vector) and a positive control (E. coliBL21 with vector containing HHDH Mzl/2G5, SEQ ID NO: 31) were included.These master well plate cultures were covered with AirPore™ microporoustape (Qiagen, Inc., Valencia, Calif.).

On day 2, the master plate cultures were gridded onto nylon membranes(Pall Biodyne B Nylon Membrane pre-cut for Omnitray, 115×76 mm, NalgeNunc #250385) then placed onto a Q-tray (Genetix USA, Inc. Beaverton,Oreg.) containing 200 ml LB agar+1% glucose, 30 μg/ml chloramphenicol.The Q-trays were incubated at 30° C. for 8-12 hours until growth wasdetected. Each nylon membrane was transferred to a Q-tray containinginducing media: 200 ml LB agar+1 mM IPTG, 30 μg/ml chloramphenicol. TheQ-trays were then incubated at 23° C. or room temperature overnight.

On day 3, the assay plate was prepared as follows: a solution of 150 mlof 10 mM Tris-SO₄, pH 7.0, and 1.0% low melt agarose was prepared andcooled to about 45° C. 5M NaCl was added to give a final concentrationof 500 mM NaCl. Bromcresol purple (BCP) and ethyl(S)-4-chloro-3-hydroxybutyrate (ECHB) were added to final concentrationsof 0.004% and 0.3%, respectively. The solution was poured into a 150 mlQ-tray and allowed to solidify.

The nylon membrane with the colonies was removed from the Q traycontaining inducing media and inverted onto the assay plate. Themembrane was imaged through the inverted Q-tray using the Alpha ImagingChemStation (Alpha Innotech Corporation, San Leandro, Calif.), aperturesetting of 4 with a 420 nm (+/−10 nm filter). An image was acquiredduring the first hour of the reaction. The intensity data for eachimaged spot was then normalized to the value of the negative controlspots. A normalized value greater than one indicated the presence ofHHDH activity. Active clones from this screen were further characterizedusing the method described in Example 5A. Clones from this screen mayalso be further characterized using the medium throughput assaydescribed in Example 5B.

B. Cyanohydrin in Agarose

This high throughput screen is used when it is desired to screen forHHDH polypeptides that exhibit HHDH activity in the presence ofcyanohydrin product, e.g., ethyl (R)-4-cyano-3-hydroxybutyrate. Theprotocol for days one and two are the same as in part A. On day 3, theassay plate was prepared as follows: a 150 ml low melt agarose solutionwas made up as follows: 10 mM Tris, pH 7.0, 2.0% low melt agarose(melted in microwave), 0.004% bromcresol purple (1.2 ml/150 ml). Thesolution was cooled to 37° C. overnight. On day three, ECHB (0.45 mlECHB/150 ml solution) and ethyl (R)-4-cyano-3-hydroxybutyrate (8.26 mlethyl (R)-4-cyano-3-hydroxybutyrate/150 ml solution) were added to givea 0.3% ECHB and 400 mM ethyl (R)-4-cyano-3-hydroxybutyrate solution. Thesolution was mixed and poured into a 150 ml Q-tray, then allowed tosolidify as described in part A.

The nylon membrane with the colonies was removed from the Q traycontaining the inducing media and inverted onto the assay plate. Themembrane was imaged as described in part A above.

Active clones from this screen were further characterized using the gaschromatography method described in Example 5B (Medium through-putassay).

Example 5 Characterization of Halohydrin Dehalogenase Activity

A. Gas Chromatography Method for Detection of ProductEthyl-(R)-4-cyano-3-hydroxybutyrate

To a solution of ethyl (S)-4-chloro-3-hydroxybutyrate (10 mM-100 mM) in500 mM HCN (500 mM NaCN adjusted to pH 7.0 with phosphoric acid) wasadded the halohydrin dehalogenase enzyme as a predissolved solution inthe same buffer. Over time, aliquots of the mixture were withdrawn andextracted with three volumes of ethyl acetate. The organic layer wasthen analysed for ethyl (R)-4-cyano-3-hydroxybutyrate by gaschromatography (GC), as described hereinbelow in Example 6. Samples weretaken at various time points, and the peak area of the productcyanohydrin, ethyl (R)-4-cyano-3-hydroxybutyrate, was plotted as afunction of time. Time points are selected at low conversion, forexample, less than 5% conversion, to avoid the effect of productinhibition (e.g., 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, etc.). The peakareas were converted to concentration units using a standard curve thatwas prepared for the ethyl (R)-4-cyano-3-hydroxybutyrate. Activity ofthe halohydrin dehalogenase was determined in units of μmol (cyanohydrinproduced)/min/mg (total halohydrin dehalogenase catalyst). Relativeactivities of some of the clones are shown in Table 2, computed asActivity of Improved HHDH Enzyme/Activity of Agrobacterium sp. HHDH (SEQID NO: 2).

TABLE 2 Relative HHDH Activity of Improved HHDH Enzymes on ECHBSubstrate Fold Improvement in HHDH Activity of Agrobacterium sp. SEQ IDNO: HHDH (SEQ ID NO: 2) (SEQ ID NO: 4) 1.5 (SEQ ID NO: 6) 1.6 (SEQ IDNO: 8) 1.8 (SEQ ID NO: 10) 1.7 (SEQ ID NO: 34) 2.4 (SEQ ID NO: 12) 2.5(SEQ ID NO: 14) 1.4 (SEQ ID NO: 16) 2.0 (SEQ ID NO: 18) 2.7 (SEQ ID NO:20) 3.8 (SEQ ID NO: 22) 2.5 (SEQ ID NO: 24) 3.2 (SEQ ID NO: 26) 1.7 (SEQID NO: 28) 2.2 (SEQ ID NO: 30) 2.8

B. Medium Throughput-Gas Chromatography Assay in Presence of CyanohydrinProduct

Hits were picked from desired wells (10 μL of culture) in the prescreenmaster well plates and transferred into the wells of 96 well NUNC plates(each well containing 200 ul LB+1% glucose, 30 μg/ml chloramphenicol(cam)) for overnight growth at 30° C., 250 rpm, 85% relative humidity.The positive controls were picked from the prescreen master well plates.

The next day, 10 μl aliquots of the overnight growth was subculturedinto 96 deep well plates each well containing 300 μl 2×YT, 100 mMNaH₂PO₄/Na₂HPO₄pH 7, 1 mM MgSO₄, 30 μg/ml cam. These plates wereincubated at 30° C., 250 rpm, 85% relative humidity, 2-4 hrs, until thecell density reached an OD 600 nm=0.6. The plates were then induced with1 mM isopropyl-13-D-thiogalactoside (IPTG) (e.g., 10 μl/well of a 34 mMIPTG stock solution or 30 uL/well of 10 mM IPTG stock) and incubated at30° C. overnight, 250 rpm, 85% relative humidity.

The next day, the plates were centrifuged (4000 rpm, 10 min., 4° C.) topellet the cells and the spent media was discarded. The plates can befrozen at −80° C. for one hour to aid in cell breakage.

The pelleted cells were lysed by adding 200 μL B-PER® lysing solution(Pierce, cat#78243) containing 2.04 M ethyl-4-cyano-3-hydroxybutyrate(“NH”) (320 g/L)(fw=157, d 1.19, 26.8 ml/100 ml lysis mixture) and 1ul/10 ml DNase (˜200 U/ul). The mixture of cells and lysing solution wasvortexed to resuspend the cells and then incubated at 50° C. withshaking for two hours.

A reaction solution was made up in a fume hood, preferably using aplastic (polypropylene) disposal container. The volume of reactionsolution was determined by number of plates screened. To prepare thereaction solution having a 1M final concentration of NaCN, NaCN(fw=49.01, 4.9 g/100 mL) was added to the desired volume of 100 mMsodium phosphate pH 7 to give 1.47M concentration of NaCN. To each 68 mLof the NaCN solution was added 24 mL of 5M stock NaCl and 8 ml ofconcentrated HCl (˜10 M) to produce the desired volume of reactionmixture that was 1.2 M NaCl, 800 mM HCl, and 1M NaCN. The final pH ofthe reaction mixture was 7.0-7.2. To this solution was added ECHB(fw=166.6, d=1.19) at 280 μL/100 mL reaction mix to obtain a 20 mM finalconcentration. The final concentrations in the reaction mix are ˜1M HCN,2M NaCl, 50 mM sodium phosphate pH 7.0 to 7.2, 20 mM ECHB.

200 μL of the reaction mixture was added to the lysed cells in eachwell. The plates were sealed using the Velocity11 PlateLoc™ heat sealer.The sealed plates were then shaken at room temperature for 120 minutes.After shaking, the plates were unsealed and 1 mL of 1 mM thymol(dissolved in ethyl acetate) was added to each well. The plates wereresealed using the Velocity11 PlateLoc™ heat sealer, shaken vigorously,then allowed to sit for ˜1 minute to let the layers separate

150 μL aliquots of the upper layer were transferred to Costar roundbottom shallow well polypropylene (PP) reaction plates (Cat# 3365) usinga Hydra™ positive displacement liquid handler (Asp mode, AV 150, AH2650, EH 37800, WH 3730, WV full, Wash 3). Samples were transferred fromthe deep well plate into the shallow well plates.

These plates were sealed using the Velocity11 PlateLoc™ heat sealer andstored at −20° C. until analysis by Gas Chromatography as described inExample 6B.

C. Medium Throughput-Gas Chromatography Assay for Inhibition in thePresence of Ethyl-4-Chloroacetoacetate (ECAA)

Hits were picked from desired wells (10 μL of culture) in the prescreenmaster well plates and transferred into the wells of 96 well NUNC plates(each well containing 200 ul LB+1% glucose, 30 μg/ml chloramphenicol(cam)) for overnight growth at 30° C., 250 rpm, 85% relative humidity.The positive controls were picked from the prescreen master well plates.

The next day, 10 μl aliquots of the overnight growth was subculturedinto 96 deep well plates, each well containing 300 μl 2×YT, 100 mMNaH₂PO₄/Na₂HPO₄pH 7, 1 mM MgSO₄, 30 μg/ml cam. These plates wereincubated at 30° C., 250 rpm, 85% relative humidity, 2-4 hrs, until thecell density reached an OD 600 nm=0.6. The plates were then induced with1 mM IPTG (e.g., 10 μl/well of a 34 mM IPTG stock solution or 30 μL/wellof 10 mM IPTG stock) and incubated at 30° C. overnight, 250 rpm, 85%relative humidity.

The next day, the plates were centrifuged (4000 rpm, 10 min., 4° C.) topellet the cells and the spent media was discarded. The plates can befrozen at −80° C. for one hour to aid in cell breakage.

The pelleted cells were lysed by adding 200 μL B-PER® lysing solution(Pierce, cat#78243) with 1 ul/10 ml DNase (˜200 U/ul). The mixture ofcells and lysing solution was vortexed to resuspend the cells and thenincubated at 50° C. with shaking for two hours.

A reaction solution was made up in a fume hood, preferably using aplastic (PP) disposal container (volume determined by number of platesscreened). To prepare the reaction solution having a 1M finalconcentration of NaCN, NaCN (fw=49.01, 4.9 g/100 mL) was added to thedesired volume of 100 mM sodium phosphate pH 7 to give 1.47Mconcentration of NaCN. To each 68 mL of the NaCN solution was added 24mL of 5M stock NaCl and 8 ml of concentrated HCl (˜10 M) to produce thedesired volume of reaction mixture that was 1.2 M NaCl, 800 mM HCl, and1M NaCN. The final pH of the reaction mixture is 7.0-7.2. To thissolution is added ECHB (fw=166.6, d=1.19) to 100 mM final concentration(1400 μL/100 mL reaction mix) and ECAA (fw=164.6, d=1.21) to 5 mM finalconcentration (100 μL/100 mL reaction mix).

200 μL of the reaction mixture was added to the lysed cells in eachwell. The plates were sealed using the Velocity11 PlateLoc™ heat sealer.The sealed plates were then shaken at room temperature for 60 minutes.After shaking, the plates were unsealed and 1 mL of 1 mM thymol(dissolved in ethyl acetate) was added to each well. The plates wereresealed using the Velocity11 PlateLoc™ heat sealer, shaken vigorously,then allowed to sit for ˜1 minute to let the layers separate

150 μl aliquots of the upper layer were transferred to Costar roundbottom shallow well polypropylene (PP) reaction plates (Cat# 3365) usinga Hydra™ positive displacement liquid handler (Asp mode, AV 150, AH2650, EH 37800, WH 3730, WV full, Wash 3). Samples were transferred fromthe deep well plate into the shallow well plates. These plates weresealed using the Velocity11 PlateLoc™ heat sealer and stored at −20° C.until analysis by Gas Chromatography as described in Example 6B.

Example 6 A. Detection of Ethyl (R)-4-cyano-3-hydroxybutyrate by GasChromotography

The ethyl (R)-4-cyano-3-hydroxybutyrate produced in Example 5A wasanalyzed using gas chromatography with flame ionization (FID) detectionusing an Agilent® HP-5™ column, 30 m long, 0.32 mm inner diameter, film0.25 μm, using the following program: 1 minute at 100° C., 5° C./minutefor 10 minutes; 25° C./minute for 2 minutes; then 2 minutes at 200° C.Inlet and outlet temperatures were both 300° C., and the flow rate was 2ml/minute. Under these conditions, ethyl (R)-4-cyano-3-hydroxybutyrateelutes at 6.25 minutes and ethyl (S)-4-chloro-3-hydroxybutyrate elutesat 4.5 minutes. Chemical purity of the species was measured using theintegrated peak areas from the gas chromoatography results.

Enantioselectivity of the halohydrin dehalogenase (HHDH) with respect toethyl (R)-4-cyano-3-hydroxybutyrate was measured by gas chromatographyand FID detection using a Restek gammaDex SA™ column (30 m long, 0.32 μminner diameter) using the following program: 25 minutes at 165° C. andflow rate at 2 ml/min. Inlet and outlet temperatures were both at 230°C. Under these conditions ethyl (R)-4-cyano-3-hydroxybutyrate elutes at19.6 minutes and ethyl (S)-4-cyano-3-hydroxybutyrate elutes at 19.2minutes.

B. Detection of Remaining Ethyl (S)-4-chloro-3-hydroxybutyrate by GasChromatography

Halohydrin dehalogenases of the present invention that exhibitedactivity in the presence of cyanohydrin product in the prescreen methodof Example 4B, were further characterized in the assay described inExample 5B. The remaining ethyl (S)-4-chloro-3-hydroxybutyrate in thereaction mixture from Example 5B was analyzed using gas chromatographywith an Agilent® 19091J-413 HP-5™ 5% phenyl methyl siloxane column, 30.0m long×320 μm inner diameter×0.25 μm nominal, and a flow rate of 2.6ml/min. The following program was used: 1 minute at 100° C., 50°C./minute for 2 minutes, 2 minutes hold, with a 10 minute cycle time.The detector conditions were as follows: 300° C., 40 ml/min H₂, 450ml/min air. Under these conditions, ethyl (S)-4-chloro-3-hydroxybutyrateelutes at 3.12 minutes, ethyl (R)-4-cyano-3-hydroxybutyrate elutes at3.06 minutes, and thymol elutes at 3.21 minutes. Activity may becharacterized by the quantity of ethyl (S)-4-chloro-3-hydroxybutyrateremaining normalized to the extraction efficiency, i.e., Area ECHB/AreaThymol. Thymol is used as an internal standard for extraction efficiencyof the reaction components from water to ethyl acetate.

Example 7 Manufacture of Ethyl (R)-4-cyano-3-hydroxybutyrate from Ethyl(S)-4-chloro-3-hydroxybutyrate

To a 3-necked jacketed 3 L flask equipped with a mechanical stirrer andconnected to an automatic titrater by a pH electrode and a feeding tubefor addition of base, was charged H₂0 (1200 mL), NaCN (37.25 g) andNaH₂PO₄ (125 g) to bring the solution to pH 7. The water circulator wasset to 40° C. After 10 minutes, halohydrin dehalogenase SEQ ID NO: 32 ascell lysate (250 mL) was added. The reaction mixture was allowed to stirfor 5 minutes. Using an addition funnel, ethyl(S)-4-chloro-3-hydroxybutyrate (45 g) was slowly added over 1 hour. ThepH was maintained at 7 by the automatic titrater by the addition of 10 MNaOH (27 mL) over 17 hours. Subsequently, gas chromatography of areaction sample showed complete conversion to product. Celite (16 g) wasadded to the flask, which was then connected to a diaphragm pump, whoseexhaust is bubbled into 5M NaOH (200 mL), to remove HCN. The mixture washeated to 60° C. under 100 mm Hg pressure. After 1 hour, a submerged airbubbler was added to the solution to aid the removal of the HCN. After 3hours, an HCN detector indicated less than 5 ppm HCN in the off-gas. Themixture was allowed to cool to room temperature, then filtered through acelite pad. The filtrate was extracted with butyl acetate (3×800 mL) andthe combined organic layers filtered through a pad of activatedcharcoal. The solvent was removed under vacuum by rotary evaporation toprovide 28.5 g of ethyl (R)-4-cyano-3-hydroxybutyrate. The purity was98% (w/w) by HPLC and the enantiomeric excess was >99% (by chiral GC,the S enantiomer was undetectable). As used herein, the term“enantiomeric excess” or “e.e.” refers to the absolute differencebetween the mole or weight fractions of major (F₍₊₎) and minor (F⁽⁻⁾)enantiomers (i.e., |F₍₊₎−F⁽⁻⁾|), where F₍₊₎+F⁽⁻⁾=1. Percent e.e. is100×|F₍₊₎−F⁽⁻⁾|. Enantiomeric composition can be readily characterizedby using the gas chromatography method described in Example 6, above,and using methods that are known in the art.

Examples 8-12 Conversion of Ethyl (R)-4-chloro-3-hydroxybutyrate toEthyl (S)-4-cyano-3-hydroxybutyrate

For each of Examples 8-12, to a 170 mL vessel connected to an automatictitrater by a pH electrode and a feeding tube for addition of base wascharged NaCN (1.5 g, 31 mmol) and water (50 mL). The vessel was sealedand the pH was adjusted to 7 by the addition of conc. H₂SO₄ (0.9 mL).The reaction mixture was heated to 40° C. and treated with a solution ofhalohydrin dehalogenase (0.4 g in 10 mL water). The halohydrindehalogenases used for these Examples had the polypeptide sequencesgiven for the following SEQ ID NOs.:

Example 8 SEQ ID No: 32 Example 9 SEQ ID No: 90 Example 10 SEQ ID No: 94Example 11 SEQ ID No: 96 Example 12 SEQ ID No: 98Then, ethyl (S)-4-chloro-3-hydroxybutyrate (5.00 g, 30.1 mmol) was addedvia syringe. The automatic titrater maintained the pH at 7 by theaddition of 4M NaCN. The progress of the reactions was monitored byrecording the cumulative volume of the NaCN solution added vs. time.

FIG. 2 shows the percent conversion of ethyl(S)-4-chloro-3-hydroxy-butyrate (calculated from the cumulativeequivalents of NaCN added) vs. time for each of these Examples. Example8 used a halohydrin dehalogenase having the amino acid sequence SEQ IDNO. 32, which is the amino acid sequence of the native halohydrindehalogenase from Agrobacterium radiobacter AD1 (hheC), expressed fromnovel nucleic acid corresponding to SEQ ID NO. 31. Comparison of thepercent conversion vs. time for Examples 9 through 12 to that of Example8 shows that novel halohydrin dehalogenases of the present inventionhave greater activity than the native halohydrin dehalogenase fromAgrobacterium radiobacter AD1 (hheC).

All publications, patents, patent applications, and other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, or other document wereindividually indicated to be incorporated by reference for all purposes.

While preferred embodiments of the invention have been illustrated anddescribed, it will be readily appreciated that various changes can bemade therein without departing from the spirit and scope of theinvention.

1. An isolated polypeptide having HHDH activity, wherein the polypeptidecomprises an amino acid sequence consisting of (a) an amino acidsequence that is at least 99% identical to SEQ ID NO: 4, 12, 16, 18, 34,38, 44, 48, 52, 66, 80, 84, 114, 154, 158, 170, or 270; (b) apolypeptide having an amino acid sequence that is at least 98% identicalto SEQ ID NO: 10, 14, or 68, 118, 164, 166, or 180; (c) an amino acidsequence that is at least 97% identical to SEQ ID NO: 110, 162, 262,422, 440 or 520; (d) an amino acid sequence that is at least 96%identical to SEQ ID NO: 116 or 448; (e) an amino acid sequence that isat least 95% identical to SEQ ID NO: 264, 266, 470 or 476; (f) an aminoacid sequence that is at least 93% identical to SEQ ID NO: 200; (g) anamino acid sequence that is at least 89% identical to SEQ ID NO: 442;(h) an amino acid sequence that is at least 88% identical to SEQ ID NO:702; (i) an amino acid sequence that is at least 80% identical to SEQ IDNO: 2, when optimally aligned with SEQ ID NO: 2, and which comprises atleast one amino acid residue selected from the group consisting of T at(residue) position 2, A or P or S at position 3, V at position 4, D atposition 6, either I or F at position 9, L at position 10, S at position13, S at position 14, K at position 15, C at position 16, T or R atposition 17, either C or S or K at position 20, T at position 24, Q atposition 26, F at position 28, T at position 29, A at position 30, L atposition 31, G at position 33, R at position 34, L at position 35, N atposition 36, H at position 37, D at position 40, L at position 44, P atposition 45, either P or A at position 47, N at position 52, V atposition 54, R at position 55, D at position 56, K at position 58, G orD at position 61, Vat position 63, R at position 72, I at position 75, Pat position 76, C at position 78, Y at position 82, either S or L atposition 84, A at position 85, E at position 91, D at position 93, Q orG at position 95, N at position 96, K at position 107, A at position112, either T, S or G at position 114, A at position 115, P at position117, N at position 120, E at position 121, P at position 122, R atposition 126, V at position 130, S at position 133, A or V at position134, L, W or V at position 136, H at position 139, I or R at position142, S at position 144, S at position 146, T at position 152, S atposition 153, either S or A at position 154, V at position 168, T atposition 169, F at position 177, V at position 178, I at position 180, Gor I at position 181, K at position 184, Y at position 186, L atposition 194, N at position 198, M at position 199, E at position 215, Gat position 236, Vat position 237, L at position 238, T at position 240,either I or A or V at position 245, Y at position 249, V or I atposition 252, and V at position 254; or (j) an amino acid sequenceencoded by a nucleic acid that hybridizes under stringent conditionsover substantially the entire length of a nucleic acid corresponding toSEQ ID NO: 1, and wherein the encoded polypeptide, when optimallyaligned with SEQ ID NO: 2, comprises an amino acid sequence having atleast one amino acid residue selected from the group consisting of T at(residue) position 2, A or P or S at position 3, V at position 4, D atposition 6, either I or F at position 9, L at position 10, S at position13, S at position 14, K at position 15, C at position 16, T or R atposition 17, either C or S or K at position 20, T at position 24, Q atposition 26, F at position 28, T at position 29, A at position 30, L atposition 31, G at position 33, R at position 34, L at position 35, N atposition 36, H at position 37, D at position 40, L at position 44, P atposition 45, either P or A at position 47, N at position 52, V atposition 54, R at position 55, D at position 56, K at position 58, G orD at position 61, V at position 63, R at position 72, I at position 75,P at position 76, C at position 78, Y at position 82, either S or L atposition 84, A at position 85, E at position 91, D at position 93, Q orG at position 95, N at position 96, K at position 107, A at position112, either T, S or G at position 114, A at position 115, P at position117, N at position 120, E at position 121, P at position 122, R atposition 126, V at position 130, S at position 133, A or V at position134, L, W or V at position 136, H at position 139, I or R at position142, S at position 144, S at position 146, T at position 152, S atposition 153, either S or A at position 154, V at position 168, T atposition 169, F at position 177, V at position 178, I at position 180, Gor I at position 181, K at position 184, Y at position 186, L atposition 194, N at position 198, M at position 199, Eat position 215, Gat position 236, V at position 237, L at position 238, T at position240, either I or A or V at position 245, Y at position 249, V or I atposition 252, and V at position
 254. 2. The isolated polypeptide ofclaim 1, wherein the polypeptide has an amino acid sequencecorresponding to SEQ ID NO: 2, but with one or more substitutionsselected from the group consisting of: S2T, either T3A or T3P, A4V, V6D,either V9I or V9F, K10L, G13S, G14S, M15K, G16C, either S17T or S17R,either R20S, R20C or R2OK, A24T, H26Q, V28F, A29T, C30A, H31L, E33G,S34R, F35L, K36N, Q37H, E40D, F44L, A45P, either T47P or T47A, K52N,M54V, S55R, E56D, E58K, either E61G or E61D, I63V, Q72R, V75I, L76P,S78C, F82Y, either P84S or P84L, E85Q, K91E, A93D, E95Q or E95G, D96N,R107K, V112A, either A114T or A114G or A114S, V115A, S117P, K120N,K121E, R122P, H126R, 1130V, A133S, T134A or T134V, F136L or F136W orF136V, W139H, L1421 or L142R, T144S, T146S, A152T, C153S, either T154Sor T154A, 1168V, P169T, Y177F, L178V, S1801, E181G or E181I, P184K,F186Y, T1941, H198N, V199M, K215E, V236G, F237V, W238L, A240T, eitherM245I or M245A or M245V, W249Y, M252V or M252I, and E254V.
 3. Theisolated polypeptide of claim 1, wherein the polypeptide has at least1.4 fold to 10,000 fold greater HHDH activity as compared to wild typeHHDH having the amino acid sequence of SEQ ID NO:
 2. 4. An isolated orrecombinant polypeptide having at least 1.4 fold greater to 10,000 foldgreater HHDH activity as compared to wild-type HHDH having the aminoacid sequence of SEQ ID NO: 2, and wherein the polypeptide is encoded bya nucleic acid that hybridizes under stringent conditions oversubstantially the entire length of a nucleic acid having a sequenceselected from the group consisting of SEQ ID NO: 3, 9, 11, 13, 15, 17,33, 37, 43, 47, 49, 51, 65, 67, 79, 83, 109, 113, 115, 117, 153, 157,161, 163, 165, 169, 179, 161, 199, 261, 263, 265, 269, 421, 439, 441,447, 469, 475, 519, 701, 725, 729, 731, 733, 735, 737, and complementarysequences thereof.
 5. An isolated or recombinant polynucleotide encodingthe polypeptide of claim
 1. 6. The isolated or recombinantpolynucleotide of claim 5, wherein the polynucleotide comprises one ormore codons selected from the group consisting of ATT encodingIsoleucine at amino acid position 5 in an encoded HHDH polypeptide; AAGencoding Lysine at amino acid position 36 in an encoded HHDHpolypeptide; ATT encoding Isoleucine at amino acid position 63 in anencoded HHDH polypeptide; GAG encoding Glutamic acid at amino acidposition 95 in an encoded HHDH polypeptide; and CCC encoding Proline atamino acid position 188 in an encoded HHDH polypeptide; wherein theamino acid position is the corresponding position in the encodedpolypeptide with reference to SEQ ID NO:
 2. 7. An expression vectorcomprising the polynucleotide of claim 5 operably linked to a promoter.8. A host cell transformed with the polynucleotide of claim
 5. 9. Amethod of making an HHDH polypeptide, said method comprising (a)cultivating the host cell of claim 8 under conditions suitable forproduction of the HHDH polypeptide, and (b) recovering the HHDHpolypeptide.
 10. The isolated polypeptide of claim 1 further havingresistance to inhibition by ECAA and the polypeptide, when aligned withSEQ ID NO: 2, having one or more of the residue changes selected fromthe group consisting of A4V, F82Y, T134V, F136W, F136V, L142R, L178V,W238L, A240T, W249Y and M252I.
 11. The polypeptide of claim 1, whereinthe polypeptide comprises an amino acid sequence that is at least 99%identical to SEQ ID NO: 4, 12, 16, 18, 34, 38, 44, 48, 52, 66, 80, 84,114, 154, 158, 170, or
 270. 12. The polypeptide of claim 1, wherein thepolypeptide comprises an amino acid sequence that is at least 98%identical to SEQ ID NO: 10, 14, or 68, 118, 164, 166, or
 180. 13. Thepolypeptide of claim 1, wherein the polypeptide comprises an amino acidsequence that is at least 80% identical to SEQ ID NO: 2, when optimallyaligned with SEQ ID NO: 2, and which comprises at least one amino acidresidue selected from the group consisting of T at (residue) position 2,A or P or S at position 3, V at position 4, D at position 6, either I orF at position 9, L at position 10, S at position 13, S at position 14, Kat position 15, C at position 16, T or R at position 17, either C or Sor K at position 20, T at position 24, Q at position 26, F at position28, T at position 29, A at position 30, L at position 31, G at position33, R at position 34, L at position 35, N at position 36, H at position37, D at position 40, L at position 44, P at position 45, either P or Aat position 47, N at position 52, V at position 54, R at position 55, Dat position 56, K at position 58, G or D at position 61, V at position63, R at position 72, I at position 75, P at position 76, C at position78, Y at position 82, either S or L at position 84, A at position 85, Eat position 91, D at position 93, Q or G at position 95, N at position96, K at position 107, A at position 112, either T, S or G at position114, A at position 115, P at position 117, N at position 120, E atposition 121, P at position 122, R at position 126, V at position 130, Sat position 133, A or V at position 134, L, W or Vat position 136, H atposition 139, I or R at position 142, S at position 144, S at position146, T at position 152, S at position 153, either S or A at position154, V at position 168, T at position 169, F at position 177, V atposition 178, I at position 180, G or I at position 181, K at position184, Y at position 186, L at position 194, N at position 198, M atposition 199, E at position 215, G at position 236, V at position 237, Lat position 238, T at position 240, either I or A or V at position 245,Y at position 249, V or I at position 252, and V at position 254.